Communication and quorum sensing in non-living mimics of eukaryotic cells.

Cells in tissues or biofilms communicate with one another through chemical and mechanical signals to coordinate collective behaviors. Non-living cell mimics provide simplified models of natural systems; however, it has remained challenging to implement communication capabilities comparable to living cells. Here we present a porous artificial cell-mimic containing a nucleus-like DNA-hydrogel compartment that is able to express and display proteins, and communicate with neighboring cell-mimics through diffusive protein signals. We show that communication between cell-mimics allows distribution of tasks, quorum sensing, and cellular differentiation according to local environment. Cell-mimics can be manufactured in large quantities, easily stored, chemically modified, and spatially organized into diffusively connected tissue-like arrangements, offering a means for studying communication in large ensembles of artificial cells

A minimal biochemical route towards de novo formation of synthetic phospholipid membranes.

All living cells consist of membrane compartments, which are mainly composed of phospholipids. Phospholipid synthesis is catalyzed by membrane-bound enzymes, which themselves require pre-existing membranes for function. Thus, the principle of membrane continuity creates a paradox when considering how the first biochemical membrane-synthesis machinery arose and has hampered efforts to develop simplified pathways for membrane generation in synthetic cells. Here, we develop a high-yielding strategy for de novo formation and growth of phospholipid membranes by repurposing a soluble enzyme FadD10 to form fatty acyl adenylates that react with amine-functionalized lysolipids to form phospholipids. Continuous supply of fresh precursors needed for lipid synthesis enables the growth of vesicles encapsulating FadD10. Using a minimal transcription/translation system, phospholipid vesicles are generated de novo in the presence of DNA encoding FadD10. Our findings suggest that alternate chemistries can produce and maintain synthetic phospholipid membranes and provides a strategy for generating membrane-based materials

Implementation and Characterization of Dynamic Genetic Networks in Vitro

Transcription and translation (TX-TL) can be performed in vitro, outside of cells, allowing the assembly and analysis of genetic networks. This approach to engineering biological networks in a less complex and more controllable environment could one day allow rapid prototyping of network designs before implementing them in living cells. Furthermore, the in vitro approach provides insight into how natural biological systems are built and is instructive to define the rules for engineering biological systems from bottom up. Despite progress in engineering TX-TL mixes for higher yields and longer synthesis times it remains challenging to implement complex genetic networks, such as oscillators, in vitro. The reason is that the reactions are usually performed in a batch format, where reaction products accumulate and synthesis rates decline over time. We addressed the problems associated with batch reactions by developing a microfluidic chip with nanoliter-scale reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these nano-reactors we can run TX-TL reactions in continuous mode keeping synthesis rates at constant steady state levels for more than 30h. The setup allows close control over the reaction conditions such as dilution rates and DNA template concentration, and to monitor mRNA and reporter protein levels in real time. We can test any genetic program of our choice just by adding the DNA templates coding for the desired functions. We implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. As a proof of concept for this reactor-based approach to engineering genetic networks we designed and implemented a novel genetic oscillator. Its network architecture consists of a positive feedback loop coupled to delayed negative feedback. Varying dilution rates and DNA template concentrations we mapped its phase diagram showing that steady state conditions were necessary to produce oscillations. The period of oscillations could be tuned by dilution rate. To demonstrate that in vitro synthetic biology is useful for prototyping of dynamic genetic networks, we compared the behavior of biomolecular ring oscillators in a cell-free framework and Escherichia coli. We implemented and characterized the ârepressilatorâ, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring the three-node and five-node oscillators to E. coli, resulting in robust and synchronized oscillations reflecting our in vitro observations. Our results demonstrate that comprehensive circuit characterization can be rapidly performed in a cell-free system and that these in vitro results have direct applicability in vivo. The reactor-based approach to in vitro synthetic biology, which was developed in this thesis, will thus enable a quantitative and more complete characterization of synthetic and natural genetic networks

Implementation of cell-free biological networks at steady state

Living cells maintain a steady state of biochemical reaction rates by exchanging energy and matter with the environment. These exchanges usually do not occur in in vitro systems, which consequently go to chemical equilibrium. This in turn has severely constrained the complexity of biological networks that can be implemented in vitro. We developed nanoliter-scale microfluidic reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these reactors we achieved transcription and translation at steady state for 30 h and implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. We constructed and implemented an in vitro genetic oscillator and mapped its phase diagram showing that steady-state conditions were necessary to produce oscillations. This reactor-based approach will allow testing of whether fundamental limits exist to in vitro network complexity

Rapid cell-free forward engineering of novel genetic ring oscillators

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the 'repressilator,' a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hours. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior

Towards a Synthetic Chloroplast

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices

Real-Time mRNA Measurement during an

In vitro transcription and translation reactions have become popular for a bottom-up approach to synthetic biology. Concentrations of the mRNA intermediate are rarely determined, although knowledge of synthesis and degradation rates could facilitate rational engineering of in vitro systems. We designed binary probes to measure mRNA dynamics during cell-free protein synthesis by fluorescence resonance energy transfer. We tested different mRNA variants and show that the location and sequence environment of the probe target sites are important parameters for probe association kinetics and output signal. Best suited for sensitive real-time quantitation of mRNA was a target site located in the 3' untranslated region, which we designed to reduce secondary structure. We used this probe target pair to refine our knowledge of mRNA dynamics in the commercially available PURE cell-free protein synthesis system and characterized the effect of TetR repressor on mRNA synthesis rates from a T7 promoter