Bridging top-down and bottom-up approaches to synthetic biology

Our research focuses on developing and applying tools for readout of cell states and for control of cellular processes, achieved via combining top-down and bottom-up approaches to synthetic biology. We use tools of protein engineering and molecular biology, together with novel synthetic cell technologies, to understand and modulate biological processes in complex systems.
We apply engineering principles to biology: we build life, or grow machines.

The top-down approach involves building on the biological ensemble, modifying existing cellular pathways to explore and control biology.
My works focuses on building gene expression quantification and control tools via engineering single strand RNA binding proteins (more).

The bottom-up approach involves building chemical systems capable of mimicking cellular processes, such as protocells with replicating RNA and minimal peptide enzymes that couple catalysis to protocell fitness.
We aim at using those systems to develop synthetic minimal cell technology, to process chemical signals between mammalian cells and the environment (more).


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bottom-up synthetic biology
Building artificial chemical systems mimicking cells

I have developed the first known system capable of non-enzymatic RNA synthesis inside model protocells.
In this work, I have shown how citric acid can stabilize fatty acid liposomes in presence of divalent cations, allowing for non-enzymatic template director RNA primer extension inside the liposomes. This discovery bridges the RNA-world hypothesis or earliest life having RNA-based metabolism with the origin of compartmentalization based on fatty acid liposomes. (Adamala and Szostak, Science 2013)
I was also involved in discovering a chemically-driven replication mechanism for model protocells (Zhu, Adamala, Zhang and Szostak, PNAS 2012).
Together, those two projects allow for drawing a plausible, complete protocell cycle: inside the protocells, RNA is replicated by template-directed non-enzymatic synthesis, and the protocells grow into filamentous threads by absorbing fatty acid molecules from micelles and divide in response to shear stress.

RNA replication inside protocells division via threads RNA is encapsulated
in protocells
activated nucleotides enter
via semi-permeable bilayer
RNA template is copied Source: Adamala and Szostak, Science 2013


As part of the same overarching goal of building a chemical system capable of Darwinian evolution, I have shown that an encapsulated small peptide catalyst can impact the fitness of model protocells.
I have studies catalysis of Ser-His and other di- and tri- amino acid catalytic peptides. I have shown that the minimal serine protease analogue, Ser-His, can catalyze formation of peptide nucleic acids (Gorlero et.al FEBS Letters 2009). I have also demonstrated how the same dipeptide catalyst's activity can be enhanced by the presence of fatty acid protocell vesicles, and in turn the product of the reaction catalyzed by said dipeptide allows the protocell to enhance uptake of membrane building blocks, resulting in protocell growth.
This couples the activity of encapsulated catalyst with the fitness of the protocell, in a model system exploring the origin of Darwinian selection. (Adamala and Szostak, Nature Chemistry 2013).

Ser-His catalysed reaction Source: Adamala and Szostak, Nature Chemistry 2013

Work done with Pier Luigi Luisi from University Roma Tre and Jack Szostak from Harvard University.


Our most significant publications in that area:

Non-enzymatic template-directed RNA synthesis inside model protocells;
K. Adamala and J.W. Szostak, Science 342 (2013) 1098 - 1100;
publisher website link
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details, abstract

Non-enzymatic template-directed RNA synthesis inside model protocells;
K. Adamala and J.W. Szostak, Science 342 (2013) 1098 - 1100;

Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg2+ on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg2+ ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg2+ - catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.


Competition between model protocells driven by an encapsulated catalyst;
K. Adamala and J.W. Szostak, Nature Chemistry 5 (2013) 495 - 501;
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details, abstract

Competition between model protocells driven by an encapsulated catalyst;
K. Adamala and J.W. Szostak, Nature Chemistry 5 (2013) 495 - 501;

The advent of Darwinian evolution required the emergence of molecular mechanisms for the heritable variation of fitness. One model for such a system involves competing protocell populations, each consisting of a replicating genetic polymer within a replicating vesicle. In this model, each genetic polymer imparts a selective advantage to its protocell by, for example, coding for a catalyst that generates a useful metabolite. Here, we report a partial model of such nascent evolutionary traits in a system that consists of fatty-acid vesicles containing a dipeptide catalyst, which catalyses the formation of a second dipeptide. The newly formed dipeptide binds to vesicle membranes, which imparts enhanced affinity for fatty acids and thus promotes vesicle growth. The catalysed dipeptide synthesis proceeds with higher efficiency in vesicles than in free solution, which further enhances fitness. Our observations suggest that, in a replicating protocell with an RNA genome, ribozyme-catalysed peptide synthesis might have been sufficient to initiate Darwinian evolution.


A simple physical mechanism enables homeostasis in primitive cells;
A. E. Engelhart*, K. Adamala*, and J. W. Szostak; Nature Chemistry, 2016, doi:10.1038/nchem.2475; *equal contribution
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publisher website link
details, abstract

Physical Basis for Homeostasis in Primordial Cells;
A. E. Engelhart*, K. Adamala*, and J. W. Szostak; Nature Chemistry, 2016, doi:10.1038/nchem.2475; *equal contribution

The emergence of homeostatic mechanisms that enabled maintenance of an intracellular steady-state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles; ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation.
This simple physical system enables a primitive homeostatic behavior: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.


Collaboration between primitive cell membranes and soluble catalysts;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, doi:10.1038/ncomms11041; *equal contribution
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details, abstract

Collaboration between primitive cell membranes and soluble catalysts;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, doi:10.1038/ncomms11041; *equal contribution

One widely-held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components.
Here, we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg2+, which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg2+ environments. The synthetic transformation requires both the catalyst and vesicles, which solubilize the water-insoluble precursor lipid.
We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells.

Whole list of peer reviewed publications: protobiology.org/publications



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Broader impact

TED talk on synthetic life
Kate will present concept of building synthetic minimal cells, with its biotechnological, biomedical and basic science implications, in a TEDx talk in November 2016, during the TEDx Beacon Street event.

Protocells in the news
The effort towards elucidating the origin and earliest evolution of life has always been receiving interest from broad audience.
As an example: a review detailing our work on RNA replication in protocells: Angewandte Chemie International Edtion Citric Acid and the RNA World 2014, 53, 5245 - 5247, external link (local copy pdf)

Our work on protocells have been featured in science news outlets and editorials, including:
Science Focus: Robert Service, The Life Force, external link (local copy pdf)
BioTechniques: How Cellular Life Evolved 06 Jan 2014 external link (local copy pdf)
Science News: To cook up life, just add citrate 185(1):15 external link (local copy pdf)
Science Daily: Researchers find missing component in effort to create primitive, synthetic cells, external link (local copy pdf)
Chemical & Engineering News: Lab-Made Protocells Show Hints Of Evolution, 91(21), 2013
The Panda's Thumb: New Szostak protocell is closest approximation to origin of life and Darwinian evolution so far, external link (local copy pdf)


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top-down synthetic biology
Engineering generalized RNA-protein interactions: a toolbox for regulation and readout of gene expression

We are developing and validating a protein architecture which binds to single stranded RNA. Using this protein technology, we are developing a tool for visualization and quantification of levels of expression of genes of interest.
This tool will work by following the reconstitution of a protein probe upon interaction of sequence-specific RNA binding proteins with the mRNA of the gene of interest.

We are also aiming to edit the transcriptome of the gene of interest, selectively decreasing the level of expression of one splice variant (as opposed to cutting the DNA of the gene, which targets all splice variants indiscriminately).
This technology could potentially help in studying non-ER translation events, elucidating mechanisms of synaptic plasticity, as well as studying healthy and diseased translational profiles of genes, e.g., those involved in oncogenesis and other disease processes.

The ability to monitor and perturb RNA in living neurons - which would open up the investigation of many processes that contribute to development, plasticity, and disease progression - would benefit greatly from a method of systematically targeting unmodified RNA sequences for observation and control. The currrent ssRNA targeting methods are based on the RNA binding protein aptamers, like MS2 or PP7; this require the introduction of aptamer binding sites into the RNA. My work shows that it is possible to develop a ssRNA binding protein that can be engineered to target arbitrary sequences of variable length, thus eliminating the need to engineer the target sites into the RNA of interest.

Popular science description: news release, local copy pdf.
This work was done with Ed Boyden

Programmable RNA-binding protein composed of repeats of a single modular unit;
Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, and Edward S. Boyden; PNAS, 2016, 10.1073/pnas.1519368113; *equal contribution
local copy pdf
publisher website link
details, abstract

Programmable RNA-binding protein composed of repeats of a single modular unit;
Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, and Edward S. Boyden; PNAS, 2016, 10.1073/pnas.1519368113; *equal contribution

The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby - RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.




©2016 kate adamala