{"id":2218,"date":"2026-06-22T21:51:51","date_gmt":"2026-06-22T21:51:51","guid":{"rendered":"http:\/\/protobiology.org\/wp3\/?p=2218"},"modified":"2026-07-01T13:51:57","modified_gmt":"2026-07-01T13:51:57","slug":"spudcell-protocols","status":"publish","type":"post","link":"http:\/\/protobiology.org\/wp3\/2026\/06\/22\/spudcell-protocols\/","title":{"rendered":"SpudCell"},"content":{"rendered":"

This work was done in collaboration with the Engelhart Lab<\/a>.\u00a0<\/p>\n

\n

What is SpudCell<\/strong><\/p>\n

SpudCell is a liposome encapsulated synthetic cell that can grow and replicate.<\/p>\n

It undergoes a full cell cycle, with DNA replication, genetically encoded feeding and genetically encoded division.\u00a0<\/p>\n<\/div>\n

SpudCell page on Biotic website<\/a> <\/p>\n

\n

Protocols for SpudCell formation and experiments<\/strong><\/span><\/p>\n

Below is the list of protocols and procedures needed to assemble and analyze a SpudCell.<\/p>\n

\n

Read this first<\/strong><\/span><\/p>\n

Synthetic cell experiments are still more resource-intensive than growing natural cells. While our motivation for this research is to make biology a general purpose technology, usable freely by all, we are currently operating in the sandbox environment.<\/p>\n

To grow a SpudCell, your lab needs to have core competencies<\/strong> in:<\/p>\n

    \n
  • liposome preparation<\/li>\n
  • PURE system expression<\/li>\n
  • aHL expression in liposomes<\/li>\n<\/ul>\n

    If you haven’t used any of those techniques before, we strongly suggest you successfully set up those first, before moving on to SpudCell experiments.<\/p>\n

     <\/p>\n

    \n

    pREP Transcription\u2013Translation and Phi29 Replication Reaction<\/strong><\/b><\/span><\/p>\n

      \n
    1. Purpose<\/strong><\/li>\n<\/ol>\n

      This protocol describes the preparation and incubation of the pREP cell-free transcription\u2013translation and DNA replication system used for protein expression, Phi29 genome replication, and related synthetic cell experiments.<\/p>\n

        \n
      1. Materials<\/strong><\/li>\n<\/ol>\n

        2.1 DNA preparation<\/strong><\/p>\n

          \n
        • Plasmid DNA (mini-prepped, Epoch 2160250)<\/li>\n
        • PCR clean-up kit (Epoch 2360250)<\/li>\n
        • Lyophilizer for DNA concentration<\/li>\n<\/ul>\n

          2.2 Commercial reagents<\/strong><\/p>\n

            \n
          • PURExpress Solution A (NEB E6800L)<\/li>\n
          • PURExpress Solution B (NEB E6800L)<\/li>\n
          • NxGen Phi29 DNA polymerase (Lucigen 30221-2)<\/li>\n
          • DTT (Goldbio DTT50)<\/li>\n
          • dNTPs (Denville Scientific CB4420-2)<\/li>\n
          • Amino acids (Sigma-Aldrich)<\/li>\n
          • Creatine phosphate (Sigma-Aldrich 27920-5G)<\/li>\n
          • Spermidine (Sigma-Aldrich S2526-5G)<\/li>\n
          • tRNA mix (E. coli<\/em>, Sigma-Aldrich 10109550001)<\/li>\n<\/ul>\n
              \n
            1. Stock Solutions (store at \u221280 \u00b0C)<\/strong><\/li>\n<\/ol>\n

              3.1 10\u00d7 Energy Mix<\/strong><\/p>\n

              Prepare and aliquot:<\/p>\n

                \n
              • 700 mM potassium glutamate<\/li>\n
              • 79 mM magnesium glutamate<\/li>\n
              • 1 M HEPES pH 8.0<\/li>\n
              • 250 mM creatine phosphate<\/li>\n
              • 3.75 mM spermidine<\/li>\n
              • 5.18 g\/L E. coli<\/em> tRNAs<\/li>\n
              • 60 mM DTT<\/li>\n
              • 3.6 mM each L-amino acid (20 standard amino acids)<\/li>\n<\/ul>\n

                3.2 50\u00d7 rNTP Mix<\/strong><\/p>\n

                Prepare and aliquot:<\/p>\n

                  \n
                • 18.75 mM ATP<\/li>\n
                • 12.5 mM GTP<\/li>\n
                • 6.25 mM CTP<\/li>\n
                • 6.25 mM UTP<\/li>\n<\/ul>\n
                    \n
                  1. Reaction Setup<\/strong><\/li>\n<\/ol>\n

                    4.1 Final reaction composition (per 100 \u00b5L reaction)<\/strong><\/p>\n

                    Prepare reactions on ice.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
                    \n

                    Component<\/strong><\/p>\n<\/td>\n

                    		\n

                    Final concentration<\/strong><\/p>\n<\/td>\n

                    		\n

                    Volume (per 100 \u00b5L)<\/strong><\/p>\n<\/td>\n<\/tr>\n<\/thead>\n

                    \n

                    10\u00d7 Energy Mix<\/p>\n<\/td>\n

                    		\n

                    1\u00d7<\/p>\n<\/td>\n

                    		\n

                    10 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    50\u00d7 rNTP Mix<\/p>\n<\/td>\n

                    		\n

                    1\u00d7<\/p>\n<\/td>\n

                    		\n

                    2 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    PURExpress Solution A<\/p>\n<\/td>\n

                    		\n

                    0.1\u00d7<\/p>\n<\/td>\n

                    		\n

                    2 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    PURExpress Solution B<\/p>\n<\/td>\n

                    		\n

                    60% v\/v<\/p>\n<\/td>\n

                    		\n

                    60 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    dATP<\/p>\n<\/td>\n

                    		\n

                    0.6 mM<\/p>\n<\/td>\n

                    		\n

                    variable (from stock)<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    dGTP<\/p>\n<\/td>\n

                    		\n

                    0.6 mM<\/p>\n<\/td>\n

                    		\n

                    variable<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    dCTP<\/p>\n<\/td>\n

                    		\n

                    0.6 mM<\/p>\n<\/td>\n

                    		\n

                    variable<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    dTTP<\/p>\n<\/td>\n

                    		\n

                    0.6 mM<\/p>\n<\/td>\n

                    		\n

                    variable<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    DTT<\/p>\n<\/td>\n

                    		\n

                    4 mM<\/p>\n<\/td>\n

                    		\n

                    variable<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    Phi29 DNA polymerase<\/p>\n<\/td>\n

                    		\n

                    0.4 U\/\u00b5L<\/p>\n<\/td>\n

                    		\n

                    variable<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    Plasmid DNA<\/p>\n<\/td>\n

                    		\n

                    as required<\/p>\n<\/td>\n

                    		\n

                    variable<\/p>\n<\/td>\n<\/tr>\n

                    \n

                    Nuclease-free water<\/p>\n<\/td>\n

                    		\n

                    \u2014<\/p>\n<\/td>\n

                    		\n

                    to 100 \u00b5L<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

                      \n
                    1. Master Mix Preparation (recommended workflow)<\/strong><\/li>\n<\/ol>\n

                      5.1 Prepare bulk reaction master mix (excluding DNA and Phi29 polymerase if variable)<\/strong><\/p>\n

                      For N reactions, prepare:<\/p>\n

                        \n
                      1. Combine in a sterile tube on ice:\n
                          \n
                        • 10\u00d7 Energy Mix (1\u00d7 final)<\/li>\n
                        • 50\u00d7 rNTP Mix (1\u00d7 final)<\/li>\n
                        • PURExpress Solution A (0.1\u00d7 final)<\/li>\n
                        • PURExpress Solution B (60% final)<\/li>\n
                        • dNTPs (0.6 mM each final)<\/li>\n
                        • DTT (4 mM final)<\/li>\n
                        • Nuclease-free water (to ~90% of final volume)<\/li>\n<\/ul>\n<\/li>\n
                        • Mix gently by pipetting (do not vortex).<\/li>\n
                        • Keep on ice until use.<\/li>\n<\/ol>\n

                          5.2 Final assembly per reaction<\/strong><\/p>\n

                          For each 100 \u00b5L reaction:<\/p>\n

                            \n
                          1. Aliquot 90\u201395 \u00b5L of master mix into PCR tubes or plate wells.<\/li>\n
                          2. Add:\n
                              \n
                            • Plasmid DNA (variable, typically 10\u2013500 ng depending on experiment)<\/li>\n
                            • Phi29 DNA polymerase to 0.4 U\/\u00b5L final concentration<\/li>\n<\/ul>\n<\/li>\n
                            • Mix gently by pipetting.<\/li>\n
                            • Briefly spin down (5\u201310 s).<\/li>\n<\/ol>\n
                                \n
                              1. Incubation<\/strong><\/li>\n<\/ol>\n
                                  \n
                                1. Incubate reactions at 30 \u00b0C<\/strong>.<\/li>\n
                                2. Standard reaction time: 8 hours<\/strong>.<\/li>\n
                                3. Avoid agitation unless specifically required for experimental variation.<\/li>\n<\/ol>\n
                                    \n
                                  1. Storage and handling notes<\/strong><\/li>\n<\/ol>\n
                                      \n
                                    • Keep all reagents on ice during setup.<\/li>\n
                                    • Avoid repeated freeze\u2013thaw cycles of energy and nucleotide mixes.<\/li>\n
                                    • Prepare aliquots of master mixes when running multiple reactions.<\/li>\n
                                    • DNA should be highly purified and concentrated prior to use.<\/li>\n<\/ul>\n

                                       <\/p>\n

                                      \n

                                      Plasmid Preparation, Cloning, and Usage for pREP \/ In Vitro Translation Experiments<\/strong><\/span><\/p>\n

                                        \n
                                      1. Purpose<\/strong><\/li>\n<\/ol>\n

                                        This protocol describes plasmid sourcing, cloning strategies, purification workflow, and concentration handling for DNA templates used in pREP transcription\u2013translation and related in vitro expression systems.<\/p>\n

                                          \n
                                        1. Plasmid Sources<\/strong><\/li>\n<\/ol>\n

                                          2.1 External plasmid sources (non-lab-cloned constructs)<\/p>\n

                                            \n
                                          • pLD1, pLD2, pLD3 plasmids (generous gift from Dr. Anthony Forster)<\/li>\n
                                          • \u03b1-hemolysin (\u03b1HL) and T7 RNA polymerase plasmids (generous gift from Dr. Vincent Noireaux)<\/li>\n<\/ul>\n

                                            2.2 Promoter system standardization<\/p>\n

                                              \n
                                            • All plasmids under T7Max promoter<\/strong> were cloned into a pre-existing lab vector system (previously described in ref. 67).<\/li>\n
                                            • This backbone contains:\n
                                                \n
                                              • UTR1 sequence (optimized for bacterial in vitro translation)<\/li>\n
                                              • T500 transcription terminator (optimized for bacterial in vitro translation)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                  \n
                                                1. Cloning Procedures<\/strong><\/li>\n<\/ol>\n

                                                  3.1 General cloning methods used
                                                  \nAll cloning was performed using one of the following:<\/p>\n

                                                    \n
                                                  • Gibson Assembly<\/strong>\n
                                                      \n
                                                    • NEB Gibson Assembly Master Mix (E2611)<\/li>\n<\/ul>\n<\/li>\n
                                                    • Site-directed mutagenesis<\/strong>\n
                                                        \n
                                                      • Q5 High-Fidelity DNA Polymerase (NEB M0493)<\/li>\n
                                                      • KLD Enzyme Mix (NEB M0554)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                          \n
                                                        1. Plasmid Amplification<\/strong><\/li>\n<\/ol>\n

                                                          4.1 Bacterial host strain<\/p>\n

                                                            \n
                                                          • DH5\u03b1 E. coli<\/em> cells used for plasmid amplification<\/li>\n<\/ul>\n

                                                            4.2 Workflow<\/p>\n

                                                              \n
                                                            1. Transform plasmid constructs into DH5\u03b1 cells<\/li>\n
                                                            2. Grow under standard antibiotic selection conditions<\/li>\n
                                                            3. Isolate plasmid DNA from overnight cultures<\/li>\n<\/ol>\n
                                                                \n
                                                              1. Plasmid Purification (Double-Clean Workflow)<\/strong><\/li>\n<\/ol>\n

                                                                5.1 Primary purification<\/p>\n

                                                                  \n
                                                                • GenCatch\u2122 Plasmid DNA Mini-Prep Kit (Epoch 2160250)<\/li>\n<\/ul>\n

                                                                  5.2 Secondary purification<\/p>\n

                                                                    \n
                                                                  • GenCatch\u2122 PCR Cleanup Kit (Epoch 2360250)<\/li>\n<\/ul>\n

                                                                    5.3 Rationale<\/p>\n

                                                                      \n
                                                                    • Double purification was used based on empirical observations (colleagues\u2019 reports) indicating improved in vitro translation yield with highly purified DNA templates.<\/li>\n<\/ul>\n
                                                                        \n
                                                                      1. DNA Quantification and Normalization<\/strong><\/li>\n<\/ol>\n

                                                                        6.1 Standard reaction concentrations<\/p>\n

                                                                          \n
                                                                        • Typical plasmid concentration in translation reactions:\n
                                                                            \n
                                                                          • 10 nM final concentration per plasmid<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                            6.2 Multi-plasmid conditions<\/p>\n

                                                                              \n
                                                                            • When multiple plasmids were used:\n
                                                                                \n
                                                                              • Each plasmid was added at equal molar concentration<\/li>\n
                                                                              • Exception:\n
                                                                                  \n
                                                                                • Whole 90 kb genome experiments used 2.5 nM per plasmid<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                    \n
                                                                                  1. DNA Handling for Multi-Plasmid Reactions<\/strong><\/li>\n<\/ol>\n

                                                                                    7.1 Problem addressed<\/p>\n

                                                                                      \n
                                                                                    • pREP reaction volume constraints result in limited free water volume after reagent addition.<\/li>\n<\/ul>\n

                                                                                      7.2 Solution for high-plasmid load (>3 plasmids)<\/p>\n

                                                                                        \n
                                                                                      1. Combine plasmid stock solutions into a single tube<\/li>\n
                                                                                      2. Lyophilize combined DNA mixture<\/li>\n
                                                                                      3. Resuspend concentrated DNA in minimal volume of nuclease-free water\n
                                                                                          \n
                                                                                        • Typical resuspension volume: ~3 \u00b5L for a 30 \u00b5L final reaction<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                          7.3 Outcome<\/p>\n

                                                                                            \n
                                                                                          • Enables high-complexity DNA inputs without increasing total reaction volume or diluting other components<\/li>\n<\/ul>\n
                                                                                              \n
                                                                                            1. Standard Use in pREP Reactions<\/strong><\/li>\n<\/ol>\n

                                                                                              8.1 DNA input integration<\/p>\n

                                                                                                \n
                                                                                              • Add plasmid DNA to final reaction mix at desired molarity (typically 10 nM per construct unless specified otherwise)<\/li>\n<\/ul>\n

                                                                                                8.2 Mixing strategy<\/p>\n

                                                                                                  \n
                                                                                                • For multi-plasmid experiments:\n
                                                                                                    \n
                                                                                                  • Pre-mix DNA stocks prior to reaction setup when necessary<\/li>\n
                                                                                                  • Ensure equal molar representation unless experimental design specifies otherwise<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                      \n
                                                                                                    1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                        \n
                                                                                                      • Store plasmid stocks at \u221220 \u00b0C<\/li>\n
                                                                                                      • Avoid repeated freeze\u2013thaw cycles<\/li>\n
                                                                                                      • Use double-purified DNA for optimal in vitro expression performance<\/li>\n
                                                                                                      • Maintain concentrated DNA stocks for multi-plasmid systems to minimize dilution effects<\/li>\n<\/ul>\n

                                                                                                         <\/p>\n

                                                                                                        \n

                                                                                                        Synthetic Cell Liposome Preparation (Oil Extrusion Method)<\/strong><\/span><\/p>\n

                                                                                                          \n
                                                                                                        1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                          This protocol describes the preparation, purification, and handling of synthetic cell liposomes used in cell-free synthetic biology experiments. Liposomes are prepared using a lipid thin-film method followed by oil hydration, emulsification, and differential centrifugation purification.<\/p>\n

                                                                                                            \n
                                                                                                          1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                            2.1 Lipids<\/strong><\/p>\n

                                                                                                              \n
                                                                                                            • DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; Avanti 850725P)<\/li>\n
                                                                                                            • DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine; Avanti 850375P)<\/li>\n
                                                                                                            • Cholesterol (Avanti 700100P)<\/li>\n<\/ul>\n

                                                                                                              2.2 Solutions<\/strong><\/p>\n

                                                                                                                \n
                                                                                                              • Chloroform (anhydrous, for lipid stocks)<\/li>\n
                                                                                                              • Mineral oil<\/li>\n
                                                                                                              • Centrifuge buffer:\n
                                                                                                                  \n
                                                                                                                • 100 mM HEPES<\/li>\n
                                                                                                                • 200 mM glucose<\/li>\n
                                                                                                                • pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                • Wash buffer:\n
                                                                                                                    \n
                                                                                                                  • 100 mM HEPES<\/li>\n
                                                                                                                  • 250 mM glucose<\/li>\n
                                                                                                                  • pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                  • External reaction buffer (as required per experiment)<\/li>\n<\/ul>\n

                                                                                                                    2.3 Equipment<\/strong><\/p>\n

                                                                                                                      \n
                                                                                                                    • Glass vials (amber preferred)<\/li>\n
                                                                                                                    • Glass syringes (for chloroform handling)<\/li>\n
                                                                                                                    • Nitrogen gas supply<\/li>\n
                                                                                                                    • Bath sonicator<\/li>\n
                                                                                                                    • Vortexer with continuous shaking attachment<\/li>\n
                                                                                                                    • Refrigerated centrifuge (4 \u00b0C capable)<\/li>\n
                                                                                                                    • Gel loading pipette tips (preferred for oil removal)<\/li>\n<\/ul>\n
                                                                                                                        \n
                                                                                                                      1. Lipid Composition<\/strong><\/li>\n<\/ol>\n

                                                                                                                        3.1 Standard formulation (\u201c1\u00d7 liposomes\u201d)<\/p>\n

                                                                                                                          \n
                                                                                                                        • Total lipid concentration: 1 mM (excluding cholesterol from total lipid calculation)<\/strong><\/li>\n
                                                                                                                        • Molar ratio (DOPE:DOPC:Cholesterol): 1:3:1<\/strong><\/li>\n<\/ul>\n

                                                                                                                          3.2 Final composition per 1 mM lipid sample<\/p>\n

                                                                                                                            \n
                                                                                                                          • DOPE: 0.25 mM<\/li>\n
                                                                                                                          • DOPC: 0.75 mM<\/li>\n
                                                                                                                          • Cholesterol: 0.25 mM<\/li>\n<\/ul>\n
                                                                                                                              \n
                                                                                                                            1. Preparation of Lipid Thin Film<\/strong><\/li>\n<\/ol>\n

                                                                                                                              4.1 Preparation of stock solutions<\/p>\n

                                                                                                                                \n
                                                                                                                              • Prepare lipid stocks in chloroform at 1 mg\/mL<\/strong><\/li>\n<\/ul>\n

                                                                                                                                4.2 Thin film formation (typical 30 \u00b5L liposome batch)<\/p>\n

                                                                                                                                  \n
                                                                                                                                1. In an amber glass vial, combine:\n
                                                                                                                                    \n
                                                                                                                                  • 5.58 \u00b5L DOPE stock<\/li>\n
                                                                                                                                  • 17.7 \u00b5L DOPC stock<\/li>\n
                                                                                                                                  • 2.9 \u00b5L cholesterol stock<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                  • Mix using glass syringes only (avoid plastic contact with chloroform).<\/li>\n
                                                                                                                                  • Evaporate chloroform under gentle nitrogen stream for 6 hours<\/strong> to form a dry lipid film.<\/li>\n
                                                                                                                                  • Cap vials tightly and store at \u221220 \u00b0C<\/strong> until use.<\/li>\n<\/ol>\n
                                                                                                                                      \n
                                                                                                                                    1. Liposome Formation (Oil Hydration and Emulsification)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                      5.1 Lipid hydration in oil phase<\/p>\n

                                                                                                                                        \n
                                                                                                                                      1. Add dried lipid film to 200 \u00b5L mineral oil<\/strong><\/li>\n
                                                                                                                                      2. Incubate at 60 \u00b0C for 3 hours<\/strong>\n
                                                                                                                                          \n
                                                                                                                                        • Vigorously shake during incubation<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                        • Sonicate in bath sonicator for 10 minutes<\/strong><\/li>\n
                                                                                                                                        • Incubate overnight at room temperature<\/strong>\n
                                                                                                                                            \n
                                                                                                                                          • Continuous vigorous shaking (vortexer with attachment)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                          • Cool mineral oil suspension to 4 \u00b0C<\/strong>\n
                                                                                                                                              \n
                                                                                                                                            • Maintain shaking during cooling<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                \n
                                                                                                                                              1. Liposome Emulsification and Encapsulation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                6.1 Emulsion formation<\/p>\n

                                                                                                                                                  \n
                                                                                                                                                1. Add 30 \u00b5L internal aqueous solution<\/strong> to mineral oil lipid suspension<\/li>\n
                                                                                                                                                2. Vortex vigorously for 5 minutes at 4 \u00b0C<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                    \n
                                                                                                                                                  1. Gradient Purification by Centrifugation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                    7.1 First centrifugation (phase separation)<\/p>\n

                                                                                                                                                      \n
                                                                                                                                                    1. Carefully layer emulsion onto:\n
                                                                                                                                                        \n
                                                                                                                                                      • 250 \u00b5L centrifuge buffer (100 mM HEPES, 200 mM glucose, pH 8)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                      • Centrifuge at:\n
                                                                                                                                                          \n
                                                                                                                                                        • 18,000 \u00d7 g (rcf), 4 \u00b0C, 15 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                        • After spin:\n
                                                                                                                                                            \n
                                                                                                                                                          • Carefully remove oil phase using gel loading tip<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                            7.2 Liposome recovery and wash<\/p>\n

                                                                                                                                                              \n
                                                                                                                                                            1. Collect liposomes from bottom aqueous phase using fresh pipette tip<\/li>\n
                                                                                                                                                            2. Transfer to:\n
                                                                                                                                                                \n
                                                                                                                                                              • 250 \u00b5L pre-chilled wash buffer (100 mM HEPES, 250 mM glucose, pH 8)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                              • Centrifuge:\n
                                                                                                                                                                  \n
                                                                                                                                                                • 12,000 \u00d7 g (rcf), 4 \u00b0C, 5 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                  7.3 Final cleanup<\/p>\n

                                                                                                                                                                    \n
                                                                                                                                                                  1. Remove any residual oil layer from top of wash buffer<\/li>\n
                                                                                                                                                                  2. Transfer liposome pellet\/phase to fresh tube<\/li>\n
                                                                                                                                                                  3. Resuspend in final external reaction buffer<\/li>\n
                                                                                                                                                                  4. Store or proceed immediately to experiments<\/li>\n<\/ol>\n
                                                                                                                                                                      \n
                                                                                                                                                                    1. Temperature Control<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                        \n
                                                                                                                                                                      • All steps from emulsification onward must be performed at 4 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                      • Maintain samples in cold room or on ice as appropriate<\/li>\n<\/ul>\n
                                                                                                                                                                          \n
                                                                                                                                                                        1. Liposome Size Control (Extrusion Parameters)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                          Liposome size is defined by extrusion membrane pore size during initial lipid processing:<\/p>\n

                                                                                                                                                                            \n
                                                                                                                                                                          • 2 \u00b5m membrane<\/strong> \u2192 synthetic cell cycle experiments<\/li>\n
                                                                                                                                                                          • 0.8 \u00b5m membrane<\/strong> \u2192 DLS characterization samples<\/li>\n
                                                                                                                                                                          • 0.4 \u00b5m membrane<\/strong> \u2192 feeder liposomes<\/li>\n<\/ul>\n
                                                                                                                                                                              \n
                                                                                                                                                                            1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                \n
                                                                                                                                                                              • Lipid stocks stored in chloroform must be kept in amber vials<\/li>\n
                                                                                                                                                                              • Thin lipid films stored at \u221220 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                              • Avoid repeated freeze\u2013thaw of lipid films<\/li>\n
                                                                                                                                                                              • Use gel-loading tips for oil removal to maximize phase precision<\/li>\n
                                                                                                                                                                              • Prevent lipid oxidation by minimizing light exposure<\/li>\n<\/ul>\n
                                                                                                                                                                                \n

                                                                                                                                                                                Fluorescent Labeling of Synthetic Cell Liposomes<\/strong><\/span><\/p>\n

                                                                                                                                                                                  \n
                                                                                                                                                                                1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                  This protocol describes incorporation of fluorescent membrane dyes into lipid thin films for preparation of labeled synthetic cell liposomes used in imaging and tracking experiments.<\/p>\n

                                                                                                                                                                                    \n
                                                                                                                                                                                  1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                    2.1 Lipid dyes (chloroform stocks)<\/strong><\/p>\n

                                                                                                                                                                                    All dyes are used as 100 \u00b5g\/mL stocks in chloroform<\/strong>:<\/p>\n

                                                                                                                                                                                      \n
                                                                                                                                                                                    • NBD-PE\n
                                                                                                                                                                                        \n
                                                                                                                                                                                      • (N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; Thermo Fisher N360)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                      • Rhodamine-PE (Lissamine\u2122 Rhodamine B PE)\n
                                                                                                                                                                                          \n
                                                                                                                                                                                        • (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; Thermo Fisher L1392)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                        • Cy5-PE\n
                                                                                                                                                                                            \n
                                                                                                                                                                                          • (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5); Avanti 810335C)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                          • Cy7-PE\n
                                                                                                                                                                                              \n
                                                                                                                                                                                            • (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7); Avanti 810337C)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                \n
                                                                                                                                                                                              1. Labeling Strategy<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                3.1 Standard dye incorporation<\/p>\n

                                                                                                                                                                                                  \n
                                                                                                                                                                                                • Dye concentration in liposomes:\n
                                                                                                                                                                                                    \n
                                                                                                                                                                                                  • 0.1 mol% per dye relative to total lipid (excluding cholesterol from total lipid calculation)<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                    3.2 Total dye loading rules<\/p>\n

                                                                                                                                                                                                      \n
                                                                                                                                                                                                    • Single dye system:\n
                                                                                                                                                                                                        \n
                                                                                                                                                                                                      • 0.1 mol% total dye<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                      • Dual dye system:\n
                                                                                                                                                                                                          \n
                                                                                                                                                                                                        • 0.1 mol% + 0.1 mol% = 0.2 mol% total dye<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                          3.3 Effective concentration example<\/p>\n

                                                                                                                                                                                                            \n
                                                                                                                                                                                                          • For 1 mM liposomes<\/strong>:\n
                                                                                                                                                                                                              \n
                                                                                                                                                                                                            • 0.1 mol% dye = 1 \u00b5M dye per lipid population<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                \n
                                                                                                                                                                                                              1. Incorporation into Lipid Thin Film<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                4.1 General procedure<\/p>\n

                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                1. During lipid thin film preparation (see liposome SOP section 4), add dye stock directly into chloroform lipid mixture.<\/li>\n
                                                                                                                                                                                                                2. Mix dye stock with lipid chloroform solutions before solvent evaporation step.<\/li>\n
                                                                                                                                                                                                                3. Proceed with nitrogen drying and thin film formation as standard.<\/li>\n<\/ol>\n
                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                  1. Dye Stock Addition (30 \u00b5L, 1 mM liposome batch)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                    For a standard 30 \u00b5L, 1 mM lipid preparation<\/strong>, add the following dye stock volumes directly into the chloroform lipid mixture:<\/p>\n\n\n\n\n\n\n\n\n
                                                                                                                                                                                                                    \n

                                                                                                                                                                                                                    Dye<\/strong><\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    Stock concentration<\/strong><\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    Volume per thin film<\/strong><\/p>\n<\/td>\n<\/tr>\n<\/thead>\n

                                                                                                                                                                                                                    \n

                                                                                                                                                                                                                    NBD-PE<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    100 \u00b5g\/mL (chloroform)<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    2.86 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                                                                                                                                                                                                                    \n

                                                                                                                                                                                                                    Rhodamine-PE<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    100 \u00b5g\/mL (chloroform)<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    4.0 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                                                                                                                                                                                                                    \n

                                                                                                                                                                                                                    Cy5-PE<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    100 \u00b5g\/mL (chloroform)<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    3.79 \u00b5L<\/p>\n<\/td>\n<\/tr>\n

                                                                                                                                                                                                                    \n

                                                                                                                                                                                                                    Cy7-PE<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    100 \u00b5g\/mL (chloroform)<\/p>\n<\/td>\n

                                                                                                                                                                                                                    		\n

                                                                                                                                                                                                                    3.98 \u00b5L<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                    1. Thin Film Formation with Dye<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                      1. Combine lipid chloroform stocks as per standard liposome preparation protocol.<\/li>\n
                                                                                                                                                                                                                      2. Add appropriate dye stock volume (from Section 5) directly into lipid chloroform mixture.<\/li>\n
                                                                                                                                                                                                                      3. Mix thoroughly using glass syringes.<\/li>\n
                                                                                                                                                                                                                      4. Proceed with:\n
                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                        • Nitrogen drying (6 hours, gentle flow)<\/li>\n
                                                                                                                                                                                                                        • Vacuum-free storage at \u221220 \u00b0C until use (sealed amber vial)<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                          1. Liposome Formation (Downstream Compatibility)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                            After dye incorporation, liposomes are prepared using the standard oil extrusion protocol:<\/p>\n

                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                            • Dye-labeled lipid films are hydrated in mineral oil<\/li>\n
                                                                                                                                                                                                                            • Follow identical emulsification, centrifugation, and purification steps as non-labeled liposomes<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                              1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                • Dye\u2013lipid mixtures in chloroform must be protected from light (use amber vials)<\/li>\n
                                                                                                                                                                                                                                • Avoid repeated freeze\u2013thaw of lipid films<\/li>\n
                                                                                                                                                                                                                                • Minimize oxygen exposure during drying to prevent dye degradation<\/li>\n
                                                                                                                                                                                                                                • Store dried thin films at \u221220 \u00b0C in dark conditions<\/strong><\/li>\n
                                                                                                                                                                                                                                • All dye handling should be performed using glass syringes where possible<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                  \n

                                                                                                                                                                                                                                  Preparation of Feeder Liposomes for Synthetic Cell Growth and Division Experiments<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                  1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                    This protocol describes the preparation of Ni-NTA\u2013functionalized feeder liposomes used in synthetic cell growth, division, and generation-tracking experiments. Feeder liposomes are designed to deliver biochemical components and enable membrane-mediated interactions such as fusion and cargo exchange.<\/p>\n

                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                    1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                      2.1 Lipids<\/strong><\/p>\n

                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                      • DOPE (Avanti 850725P)<\/li>\n
                                                                                                                                                                                                                                      • DOPC (Avanti 850375P)<\/li>\n
                                                                                                                                                                                                                                      • Cholesterol (Avanti 700100P)<\/li>\n
                                                                                                                                                                                                                                      • Ni-NTA lipid:\n
                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                        • 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel salt<\/li>\n
                                                                                                                                                                                                                                        • Avanti 790404P<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                          2.2 Lipid stock solutions<\/strong><\/p>\n

                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                          • Lipids prepared in chloroform (standard stocks)<\/li>\n
                                                                                                                                                                                                                                          • Ni-NTA lipid stock:\n
                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                            • 0.1 mg\/mL in chloroform<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                              2.3 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                              • Avanti mini-extruder (Avanti 610023)<\/li>\n
                                                                                                                                                                                                                                              • Polycarbonate membranes (0.4 \u00b5m; Avanti 610007)<\/li>\n
                                                                                                                                                                                                                                              • Orbital shaker (4 \u00b0C compatible)<\/li>\n
                                                                                                                                                                                                                                              • Cold room (4 \u00b0C)<\/li>\n
                                                                                                                                                                                                                                              • Standard liposome preparation setup (see liposome SOP section 4)<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                1. Lipid Composition<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                  3.1 Base composition (all feeder liposomes)<\/p>\n

                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                  • DOPE:DOPC:Cholesterol = 1:3:1 molar ratio<\/strong><\/li>\n
                                                                                                                                                                                                                                                  • Cholesterol is excluded from total lipid calculations<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                    3.2 Functional lipid addition<\/p>\n

                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                    • Ni-NTA lipid added at experiment-specific mol% relative to total lipid (excluding cholesterol)<\/strong><\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                      3.3 Example calculation<\/p>\n

                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                      • For 2 mM total lipid liposomes<\/strong> with 0.6 mol% Ni-NTA<\/strong>:\n
                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                        • Ni-NTA concentration = 12 \u00b5M<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                          1. Thin Film Preparation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                            4.1 Lipid mixture preparation<\/p>\n

                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                            1. Prepare lipid chloroform mixtures as per standard liposome thin film protocol.<\/li>\n
                                                                                                                                                                                                                                                            2. Add Ni-NTA lipid stock directly into chloroform lipid mixture prior to drying.<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                              4.2 Example (30 \u00b5L, 2 mM feeder liposomes, 0.6 mol% Ni-NTA)<\/p>\n

                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                              • Add 3.8 \u00b5L Ni-NTA chloroform stock (0.1 mg\/mL)<\/strong> to lipid mixture.<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                4.3 Thin film formation<\/p>\n

                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                1. Mix all lipid components in chloroform using glass syringes.<\/li>\n
                                                                                                                                                                                                                                                                2. Dry under gentle nitrogen flow to form thin film.<\/li>\n
                                                                                                                                                                                                                                                                3. Store dried film at \u221220 \u00b0C until use.<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                  1. Liposome Formation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                    5.1 Standard formation<\/p>\n

                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                    • Prepare liposomes using the standard oil extrusion method (see liposome SOP section 4).<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                      5.2 Hydration and emulsification<\/p>\n

                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                      • Hydrate lipid film in mineral oil<\/li>\n
                                                                                                                                                                                                                                                                      • Proceed with heating, sonication, and shaking steps as in standard protocol<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                        1. Liposome Extrusion<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                          6.1 Purpose<\/p>\n

                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                          • Define feeder liposome size for controlled fusion and uptake behavior<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                            6.2 Procedure<\/p>\n

                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                            1. Load liposome suspension into Avanti mini-extruder<\/li>\n
                                                                                                                                                                                                                                                                            2. Extrude through 0.4 \u00b5m polycarbonate membranes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                            3. Perform 9 extrusion passes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                            4. Collect extruded liposomes under sterile cold conditions<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                              1. Post-Extrusion Equilibration<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                1. Transfer liposomes to fresh tube<\/li>\n
                                                                                                                                                                                                                                                                                2. Incubate at 4 \u00b0C for 10 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                3. Gently shake on orbital shaker during equilibration<\/li>\n
                                                                                                                                                                                                                                                                                4. Proceed immediately to experimental use<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                  1. Functional Loading for Cell Cycle Experiments<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                    Each feeder liposome population may include:<\/p>\n

                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                    • Ni-NTA lipid: 0.6 mol% (typical value unless otherwise stated)<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                    • Generation counter oligo:\n
                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                      • 4 nM of one \u201cbottom\u201d oligonucleotide (see Methods section 12)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                      • Full pREP reaction mixture:\n
                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                        • Complete 1\u00d7 pREP system (see Methods section 2)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                          1. Standard Usage Conditions<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                            • Typical reaction volume per condition:\n
                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                              • 30 \u00b5L feeder liposomes<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                              • Lipid concentration:\n
                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                • 2 mM total lipid<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                • Used per generation in growth\/division experiments unless otherwise specified<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                  1. Temperature and Handling<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                    • All preparation and post-processing steps performed at 4 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                    • Maintain samples in cold room when possible<\/li>\n
                                                                                                                                                                                                                                                                                                    • Avoid prolonged warming after extrusion to preserve functionality<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                      1. Storage and Stability Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                        • Dried lipid films stored at \u221220 \u00b0C<\/li>\n
                                                                                                                                                                                                                                                                                                        • Ni-NTA lipid is sensitive to repeated freeze\u2013thaw cycles<\/li>\n
                                                                                                                                                                                                                                                                                                        • Extruded liposomes should be used shortly after preparation for optimal performance<\/li>\n
                                                                                                                                                                                                                                                                                                        • Avoid extended storage after loading with biochemical components<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                          \n

                                                                                                                                                                                                                                                                                                          Liposome Fusion Experiments\u00a0<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                          1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                            This protocol describes the preparation, tagging, mixing, incubation, and analysis of synthetic cell liposome fusion experiments using functionalized liposomes (e.g., protein tags, Ni-NTA systems, and \u03b1HL-mediated fusion).<\/p>\n

                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                            1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                              2.1 Liposomes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                              • Standard synthetic cell liposomes prepared via:\n
                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                • Oil extrusion method (see liposome SOP section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                • Feeder liposome protocol (see feeder SOP section 6)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                • Optional functionalized liposomes:\n
                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                  • Ni-NTA\u2013functionalized liposomes<\/li>\n
                                                                                                                                                                                                                                                                                                                  • Fluorescently labeled liposomes<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                    2.2 Fusion tags and reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                    • Fusion tag systems (prepared according to published protocols75)<\/li>\n
                                                                                                                                                                                                                                                                                                                    • \u03b1-hemolysin (\u03b1HL) system (expression-based fusion tag)<\/li>\n
                                                                                                                                                                                                                                                                                                                    • Vamp-2 fusion proteins (see dedicated section in Methods)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                      2.3 Chemicals<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                      • Triton X-100 (Acros Organics AC215682500)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                        2.4 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                        • Orbital shaker (room temperature and 30 \u00b0C capable incubator setup)<\/li>\n
                                                                                                                                                                                                                                                                                                                        • PCR tubes<\/li>\n
                                                                                                                                                                                                                                                                                                                        • 96-well plates (for endpoint fluorescence readout)<\/li>\n
                                                                                                                                                                                                                                                                                                                        • Fluorescence plate reader<\/li>\n
                                                                                                                                                                                                                                                                                                                        • Incubator set to 30 \u00b0C<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                          1. Liposome Preparation (Pre-Fusion)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                            3.1 Base preparation<\/p>\n

                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                            • Liposomes prepared as described in:\n
                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                              • Standard liposome SOP (Methods section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                              • Feeder liposome SOP (Methods section 6)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                3.2 Standard concentration<\/p>\n

                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                • All fusion samples prepared at:\n
                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                  • 1 mM total lipid concentration<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                  • Exceptions:\n
                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                    • Only as specified in experimental variations<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                      1. Fusion Tag Functionalization<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                        4.1 External fusion tag decoration<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                        1. Prepare liposomes as described above.<\/li>\n
                                                                                                                                                                                                                                                                                                                                        2. Incubate liposomes with fusion tags (per protocol75).<\/li>\n
                                                                                                                                                                                                                                                                                                                                        3. Incubation conditions:\n
                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                          • 30 minutes at room temperature<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                          • Gentle tumbling on orbital shaker<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                            4.2 \u03b1HL-based fusion system<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                            1. Prepare synthetic cells expressing \u03b1HL as described in expression protocols.<\/li>\n
                                                                                                                                                                                                                                                                                                                                            2. Incubation conditions:\n
                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                              • 3 hours at 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                              • Gentle shaking on orbital shaker inside incubator<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                1. Sample Mixing for Fusion Assays<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                  5.1 Standard mixing procedure<\/p>\n

                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                  1. After tag incubation or \u03b1HL expression:\n
                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                    • Mix liposome populations in equal volumes<\/strong>, unless otherwise specified<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                    • Each liposome population is typically:\n
                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                      • 1 mM total lipid<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                        5.2 Mixing format<\/p>\n

                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                        • Perform mixing in:\n
                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                          • PCR tubes<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                          • Use gentle pipetting to avoid mechanical rupture<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                            1. Fusion Incubation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                              6.1 Standard conditions<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                              • Incubate mixed samples:\n
                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                • 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                • Gentle shaking on orbital shaker inside incubator<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                • Standard incubation time:\n
                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                  • 6\u201312 hours<\/strong> (depending on experiment)\n
                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                    • Typical default: 12 hours<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                      1. Post-Fusion Analysis<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                        7.1 Fluorescence measurement<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                        1. Transfer samples to 96-well plates<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                        2. Measure fluorescence using plate reader<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                          7.2 Molecular analysis (if required)<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                          • For RT-PCR, qPCR, or Western blot:\n
                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                            1. Lyse liposomes using:\n
                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                              • 0.1% Triton X-100<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                              • Proceed with downstream molecular assays<\/li>\n<\/ol>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                1. Standard Reaction Conditions Summary<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                  • Lipid concentration:\n
                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                    • 1 mM total lipid per sample<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                    • Includes Ni-NTA and\/or dyes when applicable<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                    • Mixing ratio:\n
                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                      • Typically 1:1 volume ratio<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                      • Incubation temperature:\n
                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                        • 30 \u00b0C<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                        • Shaking:\n
                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                          • Gentle orbital shaking throughout incubation<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                            1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                              • Prepare fusion samples fresh when possible<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                              • Maintain gentle handling to prevent premature rupture<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                              • Avoid vigorous vortexing after fusion tag binding<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                              • Keep samples at 30 \u00b0C only during incubation phases<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                \n

                                                                                                                                                                                                                                                                                                                                                                                                FRET-Based Membrane Growth and Lipid Mixing Assay<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                  This protocol describes fluorescence resonance energy transfer (FRET)\u2013based quantification of membrane growth and lipid mixing in synthetic cell liposomes. The assay monitors dilution of membrane dyes upon liposome fusion and membrane expansion.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                  1. Principle<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                    Membrane growth is quantified by measuring dilution of FRET pairs embedded in lipid bilayers. Upon fusion with unlabeled membranes, FRET efficiency decreases, leading to increased donor fluorescence.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                    • Dye dilution \u2192 decreased FRET \u2192 increased donor signal<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                    • Signal is calibrated to % lipid mixing using defined standards<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                      1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                        3.1 Lipid dyes (FRET pairs)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                        • NBD\u2013Rhodamine pair:\n
                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                          • NBD-PE (donor)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                          • Lissamine Rhodamine-PE (acceptor)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                          • Cy5\u2013Cy7 pair:\n
                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                            • Cy5-PE (donor)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                            • Cy7-PE (acceptor)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                              3.2 Liposomes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                              • Prepared via standard liposome thin film and extrusion methods:\n
                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                • Liposome SOP (section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                • Fluorescent labeling SOP (section 5)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                  3.3 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                  • Avanti mini-extruder (Avanti 610023)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                  • 0.4 \u00b5m polycarbonate membranes (Avanti 610007)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                  • Fluorescence plate reader or spectrofluorometer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                  • Standard incubation equipment<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                    1. FRET Pair Selection Rules<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                      4.1 Pair usage<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                      • NBD\u2013Rhodamine<\/strong>\n
                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                        • Used when no GFP signal is present<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                        • Cy5\u2013Cy7<\/strong>\n
                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                          • Used when GFP is present (avoids spectral overlap with NBD)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                            1. Experimental Design<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                              5.1 Liposome populations<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                              • Population A:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                • Labeled with both donor and acceptor FRET dyes<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                • Population B:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                  • Unlabeled (no membrane dyes)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                    5.2 Mixing principle<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                    • Fusion or membrane growth leads to:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                      • Dilution of FRET dyes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                      • Decreased FRET efficiency<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                      • Increased donor fluorescence<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                        1. FRET Liposome Preparation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Prepare lipid thin films (see liposome SOP section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                          2. Incorporate FRET dyes during chloroform stage (see dye labeling SOP section 5)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                          3. Form liposomes using standard oil extrusion method<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                          4. Extrude through:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                            • 0.4 \u00b5m polycarbonate membranes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                            • Using Avanti mini-extruder<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                            • Perform standard extrusion procedure<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                              1. FRET Calibration Standards<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                7.1 Purpose<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                • Convert fluorescence signal into % lipid mixing<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                  7.2 Calibration series
                                                                                                                                                                                                                                                                                                                                                                                                                                                  \nPrepare control liposomes at defined dye dilution levels:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 0% membrane mixing<\/strong>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 1 mM liposomes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 0.1 mol% donor + 0.1 mol% acceptor (total 0.2 mol% FRET dyes)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 50% membrane mixing<\/strong>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 1 mM liposomes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 0.05 mol% donor + 0.05 mol% acceptor<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Additional calibration points prepared analogously by proportional dilution of both dyes<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                        7.3 Preparation method<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                        • All calibration samples:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Prepared from thin lipid films<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Extruded through 0.4 \u00b5m membranes (as above)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. FRET Measurement<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                              8.1 Signal acquisition<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Measure fluorescence using donor-specific wavelengths:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                • NBD or Cy5 emission channels depending on system used<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                  8.2 Spectral configuration<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Refer to Table S5 for excitation\/emission settings<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Data Interpretation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                      9.1 Signal readout<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Increased donor fluorescence indicates:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Reduced FRET efficiency<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Increased membrane mixing or growth<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                          9.2 Calibration<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Raw fluorescence values are converted to:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • % lipid mixing using calibration curve (Figure S76)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Protect dye-labeled liposomes from light<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Prepare fresh calibration standards when possible<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Avoid photobleaching during incubation and measurement<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Maintain consistent extrusion conditions across samples<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  Synthetic Cell Cycle with Feeder Liposome Growth and Mechanical Division\u00a0<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    This protocol describes a multi-generation synthetic cell cycle system combining feeder-liposome\u2013driven growth, pREP-based cell-free expression, and mechanical division via extrusion. The system supports tracking over 5 sequential generations with molecular and fluorescence-based readouts.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.1 Liposomes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Synthetic cells prepared via standard liposome protocol (Methods section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Feeder liposomes prepared via feeder protocol (Methods section 6)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Ni-NTA\u2013functionalized feeder liposomes<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2.2 Reaction components<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Complete pREP transcription\u2013translation system (Methods section 2)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Plasmid DNA templates (Methods section 3)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • T4 DNA ligase (for generation counter assembly)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Oligonucleotides for generation counter (Methods section 12)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2.3 Reagents for analysis<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Triton X-100 (0.1% final for lysis; Acros Organics AC215682500)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • EDTA (0.5 M stock, used to 10 mM final)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • DpnI restriction enzyme (NEB R0176)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • rCutSmart buffer (NEB B6004)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2.4 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Avanti mini-extruder (Avanti 610023)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 2 \u00b5m membranes (Whatman 76457-184)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Orbital shaker (30 \u00b0C incubator compatible)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Plate reader (Molecular Devices SpectraMax)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Centrifuge and lyophilizer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Heat block \/ incubator (65\u201380 \u00b0C)<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Initial Synthetic Cell Preparation (Generation 0)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                3.1 Liposome formation<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Prepare synthetic cells as described in liposome SOP (section 4)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  3.2 Initial reaction composition (per 30 \u00b5L)<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Complete 1\u00d7 pREP reaction mix (Methods section 2)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 4 \u00b5L plasmid DNA mix (Methods section 3; typically 10 nM each)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 1 \u00b5L \u201ctop strand\u201d generation counter oligos (Methods section 12)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 1 \u00b5L T4 DNA ligase<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3.3 Loading conditions<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Total internal volume: 30 \u00b5L<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • DNA templates present from start of experiment<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Generation 0 Incubation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Incubate samples:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Gentle tumbling on orbital shaker<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • This defines:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Generation 0 state<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Feeder Liposome Addition (Generation Progression)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                5.1 First growth step (Generation 1 initiation)<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Mix:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 30 \u00b5L generation 0 synthetic cells<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 30 \u00b5L feeder liposomes (2 mM total lipid)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Incubate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Gentle shaking<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Feeder Liposome Composition<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Each feeder liposome batch contains:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 2 mM total lipid concentration<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 4 nM \u201cbottom strand\u201d generation counter oligo (Methods section 12)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Complete 1\u00d7 pREP reaction mixture<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • No DNA template included<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Ni-NTA\u2013functionalized membrane lipids (Methods section 6)<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Mechanical Division (Extrusion-Based Splitting)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            7.1 End of each generation cycle<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. After 12-hour incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Extrude liposome mixture through:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 2 \u00b5m polycarbonate membrane<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Avanti mini-extruder (610023)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • This step defines completion of each generation<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Iterative Growth Cycles (Generations 1\u20135)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    For each subsequent generation:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Mix extruded population (30 \u00b5L) with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 30 \u00b5L fresh 2 mM feeder liposomes<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Incubate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Gentle shaking<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Repeat:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Extrusion (2 \u00b5m membrane)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Feeding step<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Incubation<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Analytical Workflow (Starting Generation 3)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              After each cycle (post-incubation + extrusion):<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              9.1 Fluorescence measurement<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Measure GFP fluorescence:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Plate reader (Molecular Devices SpectraMax)<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  9.2 Liposome lysis for molecular assays<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Lyse samples with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 0.1% Triton X-100 (final)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Add EDTA:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Final concentration: 10 mM<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Heat inactivate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 65 \u00b0C for 15 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          9.3 DNA digestion (new DNA quantification)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Treat with DpnI:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 5 units per reaction<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • rCutSmart buffer (1\u00d7)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Incubate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 37 \u00b0C for 30 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Heat inactivate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 80 \u00b0C for 15 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  9.4 Sample concentration and preparation<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Lyophilize samples<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2. Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 15 \u00b5L nuclease-free water<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Use for downstream analysis:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • DNA abundance<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • mRNA levels<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • generation counter RNA<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Molecular Readouts<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • DNA quantification<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • mRNA abundance<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • generation counter system output<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Control Experiments<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              11.1 Fluorescent protein control<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Experiments performed with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • GFP-expressing synthetic cells<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • GFP + mCherry competition experiments<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  11.2 No-GFP control condition<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Full 5-generation cycle repeated without GFP expression<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    11.3 Outcome comparison<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • DNA and RNA abundance compared between conditions<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Membrane dynamics validated as unchanged<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Incubation Conditions Summary<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Temperature: 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Duration per generation: 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Mixing: gentle orbital shaking<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Division: mechanical extrusion (2 \u00b5m membrane)<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Generation Counter System (Oligo Ligation and Readout)<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            This protocol describes the assembly, optimization, and quantification of a synthetic \u201cgeneration counter\u201d system based on ligation of RNA oligonucleotides with complementary sticky ends. The system is used to track synthetic cell generations in liposome-based cell cycle experiments.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2.1 Oligonucleotides<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Generation counter oligos (Table S3)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Custom synthesized RNA oligos (IDT DNA)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Used without additional purification<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2.2 Enzymes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • T4 DNA ligase (NEB M0202)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2.3 Nucleotides<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • dATP, dGTP, dCTP, dTTP (Denville Scientific CB4420-2)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2.4 Buffer components (pREP-compatible ligation conditions)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Potassium glutamate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Magnesium glutamate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • HEPES (pH 8.0)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Creatine phosphate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Spermidine<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • E. coli<\/em> tRNAs<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • DTT<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • ATP, GTP, CTP, UTP (Larova nucleotides)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.5 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Thermal incubator (30 \u00b0C)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Lyophilizer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • RT-qPCR system (Methods section 15)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Standard molecular biology pipettes and tubes<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Oligonucleotide Preparation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          3.1 Stock preparation<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Each generation counter oligo stored at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 20 nM in nuclease-free water<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3.2 Working mixture preparation<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Mix 1 \u00b5L of each 20 nM oligo stock<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2. Combine into a single oligo pool<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3. Lyophilize mixture<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              4. Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 2 \u00b5L nuclease-free water<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Use as master generation counter input<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Reaction Setup (20 \u00b5L total volume)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    4.1 Standard reaction composition<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Each 20 \u00b5L ligation reaction contains:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 1 \u00b5L T4 DNA ligase (NEB M0202)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Generation counter oligos:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 1 nM each oligo (from pooled mixture)<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • dNTPs:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 0.6 mM each of dATP, dGTP, dCTP, dTTP<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • pREP-compatible energy mix (1\u00d7 final), containing:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 70 mM potassium glutamate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 7.9 mM magnesium glutamate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.1 M HEPES pH 8.0<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 25 mM creatine phosphate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.375 mM spermidine<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.5 g\/L E. coli<\/em> tRNAs<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 10 mM DTT<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.3775 mM ATP<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.25 mM GTP<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.125 mM CTP<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.125 mM UTP<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Reaction Design Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Buffer conditions are adapted from T4 DNA ligase standard buffer (NEB B0202S), but modified to match pREP compatibility.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Non-essential transcription\u2013translation components are included to ensure compatibility with downstream synthetic cell conditions.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Reaction conditions prioritize in vivo\u2013relevant mimicry<\/em> over enzymatic optimality.<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Incubation Conditions<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Incubate ligation reactions at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 30 \u00b0C for 12 hours<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Note:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • This temperature is suboptimal for ligation efficiency<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • It is intentionally used to match synthetic cell cycle conditions<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Post-Reaction Analysis<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          7.1 RT-qPCR quantification<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Use ligation reaction as template:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 4.5 \u00b5L per RT-qPCR reaction (from 20 \u00b5L total)<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Perform RT-qPCR according to Methods section 15<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              7.2 Output measured<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Full-length generation counter ligation products<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Relative abundance across conditions<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Integration into Liposome Cell Cycle System<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  8.1 Synthetic cell (top strand loading)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Each starting \u201cparent\u201d liposome contains:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 1 nM of each \u201ctop strand\u201d oligo<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Preparation:<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Mix oligos from 20 nM stocks<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2. Lyophilize pooled mixture<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      3. Resuspend in 1 \u00b5L<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      4. Add to liposome lumen during assembly (Methods section 4)<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        8.2 Feeder liposome loading (bottom strand delivery)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Each feeder liposome contains:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 4 nM of one \u201cbottom strand\u201d oligo per generation<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Rationale:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Higher concentration compensates for dilution effects during fusion with larger synthetic cells<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Experimental Use in Cell Cycle Assays<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Generation counter system is integrated into:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Liposome growth and division experiments (Methods section 11)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Tracking:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Full-length counter product measured after:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Generation 3<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Generation 4<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Generation 5<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • RNA oligos stored at \u221220 \u00b0C (20 nM stocks)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Avoid repeated freeze\u2013thaw cycles<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Lyophilized oligo mixtures should be freshly prepared when possible<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Maintain RNase-free conditions throughout handling<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Genetically Encoded Growth and Division (FLAG-tag\u2013Mediated System)<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              This protocol describes a genetically encoded synthetic cell growth and division system using FLAG-tagged \u03b1HL, antibody\u2013streptavidin linker chemistry, azide-functionalized membrane immobilization, and feeder liposome\u2013driven growth.\u00a0<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2.1 Synthetic cells and liposomes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Synthetic cells prepared via standard liposome protocol (Methods section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Feeder liposomes prepared via feeder protocol (Methods section 6)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Ni-NTA\u2013functionalized feeder liposomes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • pREP reaction components included in feeder system<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2.2 Genetic and protein components<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Complete synthetic genome (as specified in experiment)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • FLAG-tagged \u03b1-hemolysin (\u03b1HL), 10 nM final concentration<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2.3 Immobilization chemistry<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Azido lipids (1 mol% in synthetic cell membrane; Methods section 20)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Streptavidin (5 \u00b5M stock used in reactions)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Biotin\u2013FLAG antibody linker (Abcam ab173832)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.4 Buffers and reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 100 mM HEPES, pH 8.0<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Triton X-100 (0.1% final; Acros Organics AC215682500)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2.5 Solid support<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Magnetic beads (5 mg per reaction)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2.6 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Magnetic PCR plate or magnetic rack<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Incubator with orbital shaker (30 \u00b0C)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • PCR tubes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Lyophilizer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • qPCR system (Methods section 14)<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Reagent Preparation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3.1 Biotin\u2013FLAG antibody stock<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Manufacturer stock: 1 mg\/mL (~28 \u00b5M, assuming 35 kDa)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Diluted working stock:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 20 \u00b5M in buffer<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Stored at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 4 \u00b0C<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3.2 Streptavidin working stock<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Used at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 5 \u00b5M stock<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Final concentration in reaction:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 0.1 \u00b5M<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Pre-Assembly of Linker Complex<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Mix:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Streptavidin (5 \u00b5M stock)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Biotin\u2013FLAG antibody linker (20 \u00b5M stock)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Incubate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 30 \u00b0C for 10 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Gentle mixing<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • This forms streptavidin\u2013antibody linker complex for immobilization<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Synthetic Cell Preparation (Parent Population)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    5.1 Liposome composition<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Prepared as described in liposome SOP (section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Includes:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Complete synthetic genome<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 10 nM FLAG-tagged \u03b1HL<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 1 mol% azido lipid (for immobilization)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        5.2 Immobilization setup<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Synthetic cells are immobilized onto magnetic beads prior to feeding step<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Feeder Liposome Preparation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Prepared as described in feeder liposome SOP (section 6)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Contains:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Ni-NTA lipid functionalization<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Complete pREP reaction mixture<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Immobilization and Feeding Reaction Setup<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Add to each reaction:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 5 mg magnetic beads with immobilized synthetic cells<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 30 \u00b5L feeder liposomes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 1.2 \u00b5L pre-incubated streptavidin\u2013biotin\u2013FLAG linker solution<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Generation counter configuration:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Immobilized \u201cparent\u201d cells contain:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • gen1, gen2, gen3 \u201ctop strand\u201d oligos<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Feeder liposomes contain:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • gen1, gen2, gen3 \u201cbottom strand\u201d oligos<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Detection strategy:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Full-length product detected using gen3 primers only<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Multiple oligos used to generate sufficient amplicon length for detection robustness<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Incubation Conditions<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Temperature: 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Duration: 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Mixing:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Gentle shaking in PCR tubes on orbital shaker inside incubator<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Magnetic Separation Workflow<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      9.1 Post-incubation separation<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Collect beads using magnetic PCR plate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2. Remove supernatant carefully<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      3. Wash beads with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 30 \u00b5L of 100 mM HEPES (pH 8)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Combine:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Wash solution + original supernatant fraction<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Fraction Processing<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              10.1 \u201cFree (off-beads)\u201d fraction<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Liposomes in supernatant + wash combined<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2. Lyse:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 0.1% Triton X-100<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 10 min incubation<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Lyophilize to concentrate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 5 \u00b5L water<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    10.2 \u201cBead-attached\u201d fraction<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Resuspend beads in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 50 \u00b5L 100 mM HEPES (pH 8)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Lyse attached liposomes:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 0.1% Triton X-100<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 10 min incubation<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Remove lysate from beads<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Lyophilize<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 5 \u00b5L water<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Molecular Analysis<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Analyze both fractions using qPCR:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Methods section 14<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Outputs:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Relative abundance of generation counter products<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Distribution between immobilized vs released populations<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Maintain samples at 4 \u00b0C before incubation<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Avoid bead drying prior to lysis steps<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Handle azido-functionalized liposomes carefully to preserve surface chemistry<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Perform all post-lysis concentration steps promptly<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Western Blot Analysis of Protein Expression<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          This protocol describes SDS-PAGE and Western blot analysis of proteins expressed in solution or within synthetic cell liposomes, including sample preparation, electrophoresis, transfer, immunodetection, and chemiluminescent imaging.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2.1 Samples<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Cell-free expression reactions in solution<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Synthetic cell liposome samples (Methods section 4 and related protocols)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2.2 Lysis reagent (for liposome samples)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Triton X-100 (0.1% final concentration)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2.3 SDS-PAGE reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 2\u00d7 SDS loading buffer:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 100 mM Tris-HCl<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 2.5% SDS<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 20% glycerol<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 4% \u03b2-mercaptoethanol<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 0.1% bromophenol blue<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Acrylamide:Bis-acrylamide gel (37.5:1)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • SDS running buffer:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 25 mM Tris<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 192 mM glycine<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 3.5 mM SDS<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.4 Transfer system<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Nitrocellulose membrane (0.2 \u00b5m)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Transfer buffer:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 25 mM Tris<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 192 mM glycine<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2.5 Immunoblotting reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • TBST buffer:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 20 mM Tris (pH 7.4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 150 mM NaCl<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 0.05% Tween-20<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Nonfat dry milk (blocking reagent, 5%)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Mouse IgG1 anti-His primary antibody (BioLegend 652505)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • HRP-conjugated goat anti-mouse IgG1 secondary antibody (BioLegend 405306)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2.6 Detection system<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • SuperSignal chemiluminescent substrate (Thermo Scientific 34577)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2.7 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Mini-PROTEAN electrophoresis system (Bio-Rad)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • PowerPac 3000 power supply (Bio-Rad)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Thermocycler or heat block (95 \u00b0C capability)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Horizontal rocker<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • ChemiDoc MP imaging system (Bio-Rad)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Image Lab software v5.2.1<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Sample Preparation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3.1 Solution samples (no vesicles)<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Mix directly with loading buffer (1:1 ratio with 2\u00d7 SDS loading buffer)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      3.2 Liposome samples<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Lyse samples with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 0.1% Triton X-100<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Proceed to mixing with loading buffer (1:1 with 2\u00d7 SDS loading buffer)<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Denaturation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Incubate samples in loading buffer at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 95 \u00b0C for 5 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Use thermocycler or heat block<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. SDS-PAGE Electrophoresis<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Load samples onto:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 37.5:1 acrylamide:bis-acrylamide gels<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Run gel using Mini-PROTEAN system<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Conditions:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 100 V for 60 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • In 800 mL SDS running buffer<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Protein Transfer<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Transfer proteins to:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 0.2 \u00b5m nitrocellulose membrane<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Conditions:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 100 V for 60 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • In 1 L transfer buffer<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Membrane Blocking<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Incubate membrane in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 5% nonfat milk in TBST<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Conditions:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 60 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Horizontal rocking<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Primary Antibody Incubation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Prepare antibody solution:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Mouse IgG1 anti-His antibody (BioLegend 652505)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Dilution: 1:5000 in 5% milk\/TBST<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Incubate membrane:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 60 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Horizontal rocker<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Washing Steps<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Wash membrane:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 3\u00d7 quick rinses with TBST<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 3\u00d7 10-minute washes with TBST<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Secondary Antibody Incubation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Prepare solution:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • HRP-conjugated goat anti-mouse IgG1 (BioLegend 405306)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Dilution: 1:5000 in 5% milk\/TBST<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Incubate membrane:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 60 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Horizontal rocking<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Final Washes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Repeat washing:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 3\u00d7 TBST rinses<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 3\u00d7 10-minute TBST washes<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Chemiluminescent Detection<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Apply:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • SuperSignal substrate (Thermo Scientific 34577)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Develop blot according to manufacturer instructions<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Imaging<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Image membrane using:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • ChemiDoc MP Imaging System (Bio-Rad)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Software:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Image Lab v5.2.1<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Keep membranes hydrated during all antibody steps<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Avoid drying of nitrocellulose membrane before detection<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Prepare antibody dilutions fresh for optimal signal quality<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Ensure complete removal of Triton X-100 prior to electrophoresis<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Size Exclusion Chromatography (SEC) Purification of Liposomes<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        This protocol describes gravity-driven size exclusion chromatography (SEC) used to separate liposomes from unencapsulated solutes following synthetic cell or liposome preparation.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2.1 Chromatography column<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Poly-Prep Chromatography Column (Bio-Rad 7311550; 10 mL plastic column)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2.2 Size exclusion resin<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Sepharose 4B beads (Sigma-Aldrich 4B200-1L)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Bead size: 45\u2013165 \u00b5m<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Stored as ethanol slurry<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2.3 Buffers<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 50 mM HEPES, pH 8.0<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2.4 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Fraction collector (Gilson FC203B)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 96-well collection plates<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Gravity flow setup<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Column Packing<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Fill 10 mL chromatography column with Sepharose 4B ethanol slurry.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2. Allow beads to settle by gravity.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3. Drain ethanol solvent completely.<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Column Equilibration<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Wash column with \u22654 column volumes<\/strong> of:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 50 mM HEPES, pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Avoid disturbing the bead bed during washing.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • After final wash:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Drain buffer until liquid level is just above bead surface.<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Sample Loading<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Prepare liposome sample (typically 20\u2013100 \u00b5L).<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2. Gently apply sample dropwise across the entire column surface.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                3. Minimum effective loading volume:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 20 \u00b5L<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Prevent disruption of bead surface during loading.<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Elution<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Immediately add 50 mM HEPES buffer to cover bead surface after loading.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2. Ensure column does not dry out at any point.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      3. Allow elution to proceed by gravity flow only<\/strong>.\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Do not apply pressure or accelerate flow.<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Fraction Collection<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Collect eluate into 96-well plates using:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Gilson FC203B fraction collector<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Collect at least:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 48 fractions per column run<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Ensure full separation of:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Liposome-containing fractions<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Free (unencapsulated) solute fractions<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Post-Purification Processing<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Analyze fractions using plate reader (initial screening).<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2. Identify liposome-containing fractions based on signal profiles.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      3. If required:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Recover selected fractions from wells<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Concentrate via lyophilization<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Perform downstream biochemical or fluorescence assays<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Critical Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Do not disturb bead bed at any stage after equilibration<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Maintain continuous buffer coverage to prevent column drying<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Apply samples gently to avoid mixing with resin surface<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Use gravity flow exclusively for reproducible separation<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Single-Cell Plasmid and Generation Counter Product Abundance Analysis<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                This protocol describes isolation, dilution, and molecular screening of single synthetic cells to quantify plasmid distribution and generation counter products after growth and division cycles.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2.1 Synthetic cells<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Synthetic cell populations after growth\/division experiments<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Feeder liposome removal system (dialysis; Methods section 24)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2.2 Molecular biology reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 16S rRNA primers (Table S2)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • dNTPs (Denville Scientific CB4420-2)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Superscript IV Reverse Transcriptase (Invitrogen 18090010)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Superscript IV RT buffer (5\u00d7)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • RNase Inhibitor Murine (NEB M0314S)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • OneTaq 2\u00d7 Master Mix (NEB M0482)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Chai Green Dye (Chai R01200S)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.3 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Cold room (4 \u00b0C)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Thermocycler \/ heat blocks (52\u201380 \u00b0C capability)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • qPCR systems:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Chai Open qPCR machine (single or dual channel)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • CFX Opus 384 System<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • \u221280 \u00b0C storage<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 384-well PCR workflow setup<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Sample Preparation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            3.1 Removal of feeder liposomes<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Dialyze synthetic cell samples to remove feeder liposomes (see Methods section 24).<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              3.2 Normalization<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Adjust sample concentration to:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • OD ~0.2 equivalent synthetic cell concentration<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  3.3 Single-cell dilution strategy<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Perform sequential dilution at 4 \u00b0C (cold room)<\/strong>.<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2. Work rapidly to minimize leakage near critical aggregation concentration (CAC).<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  3. Target dilution:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • ~1 synthetic cell per 1 \u00b5L<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Collect:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 0.8 \u00b5L per aliquot<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Total: 384 aliquots<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Single-Cell Occupancy Verification<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          4.1 Rationale<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Many aliquots are expected to be empty<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Presence of ribosomes (16S rRNA) used as proxy for cell presence<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            4.2 Screening marker<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • E. coli 16S rRNA amplification used to confirm cell-containing wells<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Reverse Transcription (RT) Setup (10 \u00b5L reaction)<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                For each putative single-cell aliquot:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Prepare initial mix:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 0.8 \u00b5L sample<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 0.5 \u00b5L 2 \u00b5M forward 16S primer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 0.5 \u00b5L 10 mM dNTPs<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 4.7 \u00b5L nuclease-free water<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 65 \u00b0C for 1 minute<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Immediately place on ice for \u22655 minutes<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Add RT components:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 2 \u00b5L 5\u00d7 Superscript IV buffer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 0.5 \u00b5L 100 mM DTT<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 0.5 \u00b5L Superscript IV RT (200 U\/\u00b5L)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 0.5 \u00b5L RNase inhibitor (40 U\/\u00b5L)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • RT incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 52 \u00b0C for 10 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Enzyme inactivation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 80 \u00b0C for 10 minutes<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Storage:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Retain remaining 8 \u00b5L at \u221280 \u00b0C<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. PCR Amplification (OneTaq)<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Prepare 20 \u00b5L PCR reaction:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 2 \u00b5L RT product (template)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • OneTaq 2\u00d7 Master Mix (NEB M0482)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Chai Green Dye (1\u00d7 final concentration)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Run PCR following manufacturer-recommended cycling conditions.<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. qPCR and Detection<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Analyze amplification using:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Chai Open qPCR system (single or dual channel), or<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • CFX Opus 384 System<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Determine:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Presence\/absence of synthetic cell per aliquot (via 16S signal)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Positive wells selected for downstream plasmid and generation counter analysis<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Downstream Interpretation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              8.1 Cell-containing selection<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Only 16S-positive wells are considered valid single-cell samples<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                8.2 Quantification targets<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Plasmid copy distribution<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Generation counter product abundance<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Critical Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Maintain all dilution steps at 4 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Minimize handling time during serial dilution<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Avoid membrane leakage by rapid processing near CAC conditions<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Expect high fraction of empty wells in 384-well sampling scheme<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Store intermediate RT products at \u221280 \u00b0C when needed<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Genetically Encoded Division of Synthetic Cells (Streptavidin\u2013Biotin NTA System)<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        This protocol describes a genetically encoded division system for synthetic cells using streptavidin\u2013biotin\u2013NTA crosslinking to induce membrane organization and division-like behavior. The system supports both free-solution and immobilized bead-based formats, including competitive growth experiments.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2.1 Proteins and linkers<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Streptavidin (BioLegend 280302; 1.0 mg\/mL ~19 \u00b5M stock)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Biotin-NTA linker (VWR 90074)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2.2 Buffers<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 50 mM HEPES, pH 8.0<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 100 mM HEPES, pH 8.0<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • PBS<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2.3 Liposomes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Synthetic cells prepared as in cell cycle SOP (Methods section 11)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Feeder liposomes (when applicable, excluded from division system)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Liposomes without generation counter oligos (for division-only experiments)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2.4 Solid support<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Magnetic beads (5 mg per sample)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Azide-blocked immobilization surface (when used)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2.5 Molecular biology reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Triton X-100 (0.1%; Acros Organics AC215682500)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • qPCR reagents (Methods section 14)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    2.6 DNA markers (competitive experiments only)<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • CMV-GFP plasmid (10 nM encapsulated)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • CMV-mCherry plasmid (10 nM encapsulated)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.7 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Orbital shaker (30 \u00b0C incubator compatible)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • PCR tubes<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Magnetic PCR rack\/plate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • DLS instrument (Methods section 19)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • qPCR system<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Reagent Preparation<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          3.1 Streptavidin working stock<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Start from:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 1.0 mg\/mL (~19 \u00b5M) stock<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Dilute in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 50 mM HEPES pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Final working concentration:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 5 \u00b5M<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Store at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 4 \u00b0C<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3.2 Biotin-NTA linker working stock<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 100 mM HEPES pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Final concentration:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 20 \u00b5M<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Store at:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 4 \u00b0C<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Stoichiometric Design (Binding Optimization)<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Liposome concentration reference:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • ~6.71 \u00d7 10\u00b9\u2070 liposomes per mL at 1 nM (1 \u00b5m diameter)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • \u2248 0.003 pmol liposomes per 30 \u00b5L sample<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Target loading:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • ~1000 streptavidin molecules per liposome<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Final working concentrations:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Streptavidin: 0.1 \u00b5M (3 pmol per 30 \u00b5L)<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Biotin-NTA linker: 0.4 \u00b5M (4\u00d7 excess relative to streptavidin binding sites)<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Pre-Complex Formation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Mix per sample:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.6 \u00b5L of 5 \u00b5M streptavidin stock<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 0.6 \u00b5L of 20 \u00b5M biotin-NTA linker stock<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 30 \u00b0C for 10 minutes<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Gentle mixing<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Synthetic Cell Preparation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Liposomes prepared as in cell cycle SOP (Methods section 11)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • For division-only experiments:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • No generation counter oligos included<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    PART A \u2014 Free-Solution Division Experiments<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    7A. Reaction Setup<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Combine:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 30 \u00b5L synthetic cells (1 mM total lipid)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 1.2 \u00b5L pre-incubated streptavidin\u2013biotin-NTA complex<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        8A. Incubation<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Conditions:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 30 \u00b0C<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Gentle shaking in PCR tubes<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            9A. Post-Incubation Processing<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Dilute samples to:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 0.1 mM total lipid in PBS<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Analyze by:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Dynamic light scattering (DLS; Methods section 19)<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  PART B \u2014 Immobilized Division Experiments<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  7B. Bead Immobilization Setup<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Combine:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 5 mg magnetic beads with immobilized 30 \u00b5L synthetic cells (azide-blocked)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 1.2 \u00b5L streptavidin\u2013biotin-NTA pre-complex<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      8B. Incubation<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 30 \u00b0C for 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Gentle shaking in PCR tubes<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        9B. Fraction Separation<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Magnetically separate beads<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2. Collect supernatant (\u201cdaughter fraction\u201d)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        3. Wash beads with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 50 \u00b5L 100 mM HEPES pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Combine:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Wash + original supernatant \u2192 daughter fraction<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              10B. Fraction Processing<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              10B.1 Daughter fraction<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Lyse with:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 0.1% Triton X-100 (10 min)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Lyophilize<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 5 \u00b5L water<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    10B.2 Bead-bound fraction<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Resuspend beads in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 100 \u00b5L 100 mM HEPES pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Lyse:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 0.1% Triton X-100 (10 min)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Remove lysate<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Lyophilize<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 5 \u00b5L water<\/strong><\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Analysis<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Quantify both fractions using qPCR (Methods section 14)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Outputs:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • Distribution of synthetic cell material between daughter and retained populations<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  PART C \u2014 Competitive Growth Experiments<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  12C. System Setup<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Prepare two bead-immobilized populations:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • T7Max \u03b1HL synthetic cells<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • T7 \u03b1HL synthetic cells<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Each population:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 5 mg beads + 30 \u00b5L synthetic cells<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        13C. Genetic Markers<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        To distinguish populations:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • T7 cells:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 10 nM CMV-GFP plasmid<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • T7Max cells:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • 10 nM CMV-mCherry plasmid<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Note:<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • CMV promoter ensures no bacterial expression burden<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Used solely as neutral lineage markers<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                14C. Competition Reaction<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Mix both populations<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2. Add streptavidin\u2013biotin-NTA complex as above<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                3. Incubate:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 30 \u00b0C for 12 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Gentle shaking<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    15C. Post-Reaction Processing<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Same bead separation and fraction workflow as Section 9B<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Wash and resuspension volumes doubled relative to single-population experiments<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Analyze daughter fraction via qPCR and marker plasmid readout<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Storage and Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Maintain protein reagents at 4 \u00b0C<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Avoid prolonged incubation of streptavidin\u2013biotin complexes before use<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Handle beads gently to prevent loss of immobilized synthetic cells<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Keep liposome samples at 30 \u00b0C only during active incubation steps<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Immobilization of Synthetic Cells on Magnetic Beads (DBCO\u2013Azide Coupling)<\/strong><\/span><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Purpose<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            This protocol describes covalent immobilization of azide-functionalized synthetic cell liposomes onto DBCO-functionalized magnetic beads via click chemistry, including bead blocking to prevent post-division re-binding of daughter vesicles.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Materials<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              2.1 Lipids and liposomes<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Synthetic cells prepared as in standard liposome protocol (Methods section 4)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 16:0 azidocaproyl PE (Avanti 870126P)\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • 1 mol% incorporated into liposome membranes<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2.2 Magnetic beads<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • DBCO-tagged magnetic beads (Kerafast FCC433)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Binding capacity:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 30\u201350 nmol DBCO per mg beads<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.3 Blocking reagent<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 3-Azido-L-alanine (Jena CLK-AA003)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Prepared in 50 mM HEPES pH 8.0 (50 mM stock)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        2.4 Buffers and lysis reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • 50 mM HEPES, pH 8.0<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Triton X-100 (0.1%; Acros Organics AC215682500)<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          2.5 Molecular biology reagents<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • qPCR reagents targeting \u03b1HL plasmid<\/li>\n<\/ul>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            2.6 Equipment<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Magnetic rack \/ PCR tube magnetic stand<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Incubator at 30 \u00b0C<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Shaker for gentle mixing<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Lyophilizer<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • qPCR system<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Preparation of Azide-Functionalized Liposomes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Prepare liposomes using standard protocol (Methods section 4).<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2. Include:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • 1 mol% 16:0 azidocaproyl PE<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Stock preparation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 0.1 mg\/mL azido lipid stock in chloroform<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Add:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 2.54 \u00b5L per 30 \u00b5L sample (1 mM liposomes)<\/strong> during thin film formation<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Final azide concentration:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • ~10 \u00b5M per 30 \u00b5L sample<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Bead Preparation<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Wash DBCO beads:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • 3\u00d7 with 50 mM HEPES pH 8.0<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Estimate functional groups:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                • ~250 nmol DBCO in 5 mg beads<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Liposome\u2013Bead Coupling Reaction<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Combine per reaction:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 5 mg DBCO magnetic beads<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 30 \u00b5L azide-labeled liposomes<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Reaction stoichiometry:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Liposomes: ~0.3 nmol azide total<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Note: ~50% accessible due to bilayer leaflet distribution<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Large excess of DBCO ensures complete capture<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 30 \u00b0C for 8 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Gentle shaking<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • During incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Synthetic cell internal reactions (e.g., translation) may be initiated simultaneously if required<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1. Immobilization Verification (Control Assay)<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Magnetically separate beads<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                2. Collect:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Supernatant (\u201cfree fraction\u201d)<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Lyse remaining bead-bound liposomes:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Add 20 \u00b5L:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 50 mM HEPES pH 8.0<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 0.1% Triton X-100<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Incubate to ensure complete lysis<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Process both fractions:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Lyophilize<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Resuspend in:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • 2 \u00b5L 50 mM HEPES pH 8.0<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          • Analyze via qPCR:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Target: \u03b1HL plasmid<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • Expected result:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Bead fraction: full recovery of \u03b1HL plasmid (\u224810 nM equivalent input)<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Supernatant: no detectable \u03b1HL signal<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. DBCO Blocking to Prevent Rebinding of Daughter Cells<\/strong><\/li>\n<\/ol>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  7.1 Rationale<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  Excess DBCO groups remain on beads after immobilization and may capture newly formed daughter liposomes, interfering with division experiments.<\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  7.2 Blocking reaction<\/strong><\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. After 8-hour immobilization step, add:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • 7 \u00b5L of 50 mM 3-Azido-L-alanine solution<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Final azide added: 350 nmol<\/strong><\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    • Incubation:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 4 hours<\/strong><\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • 30 \u00b0C<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Gentle shaking<\/li>\n<\/ul>\n<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      • Outcome:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        • Unreacted DBCO sites are quenched via azide coupling<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          1. Post-Blocking Usage<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            • After blocking, bead\u2013liposome complexes are considered ready for:\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Division experiments<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Growth and feeder liposome assays<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              • Competitive population studies<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                1. Critical Handling Notes<\/strong><\/li>\n<\/ol>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Maintain excess DBCO-to-azide ratio for full immobilization<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Ensure complete bead washing prior to coupling<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Avoid bead aggregation during incubation<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Always perform blocking step before division experiments<\/li>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  • Handle azide compounds carefully due to reactivity<\/li>\n<\/ul>\n
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    \n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     <\/p>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     <\/p>\n<\/div>\n

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     <\/p>\n","protected":false},"excerpt":{"rendered":"

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    This work was done in collaboration with the Engelhart Lab.\u00a0 SpudCell page on Biotic website  <\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[11],"tags":[],"class_list":["post-2218","post","type-post","status-publish","format-standard","hentry","category-static"],"_links":{"self":[{"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/posts\/2218","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/comments?post=2218"}],"version-history":[{"count":22,"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/posts\/2218\/revisions"}],"predecessor-version":[{"id":2325,"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/posts\/2218\/revisions\/2325"}],"wp:attachment":[{"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/media?parent=2218"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/categories?post=2218"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/protobiology.org\/wp3\/wp-json\/wp\/v2\/tags?post=2218"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}