(142d) Developing Robust, Environmentally-Responsive Genetic Circuits for Real-World Applications

Moon, T. S., Washington University in St. Louis
Shopera, T., Washington University in St. Louis
Hoynes-O'Connor, A., Washington University in St. Louis
Lee, Y. J., Washington University in St. Louis
Rottinghaus, A., Washington University in St. Louis
As synthetic biology transitions out of the lab and towards real-world applications, there is a need to develop a well-characterized toolbox of genetic sensors and regulators that can respond to extrinsic environmental conditions, instead of lab chemicals (e.g., IPTG). In addition, many sophisticated cellular functions require complex genetic circuits, which can lead to significant consumption of finite cellular resources and thus resource-coupled interference in predictable circuit behavior. To solve these problems, we have developed environmental sensors, RNA regulators, and feedback circuits. First, we have built and characterized sensors that respond to pH, temperature, light, oxygen levels, nitrogen source levels, or concentrations of diverse compounds found in a variety of environments. These environmental sensors have also been combined to construct multi-input logic gates (e.g., AND gates). Second, we have employed the CRISPR interference system (CRISPRi) and synthetic antisense RNAs (asRNAs) to repress or derepress target genes in a programmable manner. Importantly, we provided design rules for asRNA regulators, based on a comprehensive study of more than a hundred de novo designed asRNA regulators, enabling predictable implementation of these regulators in complex genetic circuits (e.g., NAND gates that respond to pH and temperature). Third, we have investigated the effects of resource competition on circuit behaviors. For the first time, we experimentally demonstrated that negative feedback can significantly reduce resource-coupled interference in complex circuits, allowing for robust circuit performance. These environmental sensors, RNA regulators, and feedback motif lay the groundwork for the implementation of robust, environmentally-responsive genetic circuits in metabolic engineering, environmental, and medical applications.