Development and Characterization of Genetic Sensors and Regulators for the Construction of Environmentally-Responsive Genetic Circuits

Genetic circuits enable engineers to program complex logical behaviors into living organisms. Organisms can be programmed to optimize the production of fuels and chemicals, diagnose and treat diseases, or remediate environmental pollutants. A well-characterized toolbox of genetic sensors and regulators is needed to construct these circuits. Genetic sensors that respond to environmentally-relevant signals allow circuits to evaluate the cell\u27s conditions, and versatile and designable regulators translate information about the cell\u27s environment into the desired response. In this work, we demonstrate the de novo design of RNA thermosensors in Escherichia coli, and integrate these sensors into complex genetic circuits. Next, we provide a large-scale analysis of antisense RNA regulators, generate design rules for these regulators, and validate these design rules through the construction of genetic circuits with predictable behaviors. Finally AND and NAND gates are developed that respond to temperature and pH, and utilize protein and RNA regulators. The sensors, regulators, and circuits developed and characterized here represent a substantial contribution to the synthetic biology toolbox. Furthermore, this work constitutes an important step forward in enabling genetic circuits to overcome challenges in chemical synthesis, medicine, and environmental remediation

De novo design of heat-repressible RNA thermosensors in E-coli

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a hairpin structure at lower temperatures. Here, we demonstrate the de novo design of short, heat-repressible RNA thermosensors. These thermosensors contain a cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5′ untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem–loop, and gene expression is unobstructed. At high temperatures, the stem–loop unfolds, allowing for mRNA degradation and turning off expression. We demonstrated that these thermosensors respond specifically to temperature and provided experimental support for the central role of RNase E in the mechanism. We also demonstrated the modularity of these RNA thermosensors by constructing a three-input composite circuit that utilizes transcriptional, post-transcriptional, and post-translational regulation. A thorough analysis of the 24 thermosensors allowed for the development of design guidelines for systematic construction of similar thermosensors in future applications. These short, modular RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications