A synthetic gene network architecture that propagates
Afeyan, Raffi Badrig
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Synthetic biology is a field that is tending towards maturity. Synthetic gene networks are becoming increasingly more complex, and are being engineered with functional outcomes as design goals rather than just logical demonstration. As complex as circuits become, it is still a difficult process to build a functional gene network. Much work has been done to reduce DNA assembly time, but none specifically addresses the complexity ofproducing functional networks. To this end, we present a synthetic gene network assembly strategy that emphasizes characterization-driven iteration. The Plug- and-Play methodology allows for post-construction modification to circuits, which enables the simple swapping of parts. This type of modification makes it possible to tune circuits for troubleshooting, or even to repurpose networks. We used a specified set of restriction enzymes, a library of optimized parts and a compatible backbone vector system to preserve uniqueness of cloning sites and allow maintained post-construction access to the network. To demonstrate the system, we rapidly constructed a bistable genetic toggle and subsequently transformed it into two functionally distinct networks, a 3 and 4-node feed-forward loop. We also designed a synthetic gene network that can propagate signals across a population ofisogenic bacteria. We used the Plug-and-Play methodology to quickly construct an excitable system that toggles between sending and receiving states. We developed a spatial assay platform that could accommodate long-term, large-scale plating experiments so as to visualize the propagation effect on the centimeter scale. We built several iterations ofthe propagating network, probed the regulatory dynamics ofthe various nodes and identified problematic nodes. We took steps to address these nodes with both orthogonal transcription machinery as well as multiple modes of genetic regulation. We integrated the propagating networks with a DNA-damage sensitive triggering module. This opened up the gene network to potentially complex applications such as antibiotic sensing, or longer-distance communication experiments.
Thesis (Ph.D.)--Boston University