(59e) Development of Heat-Repressible RNA Thermosensors in Bacteria

Inducible gene expression systems are powerful tools for optimizing metabolic pathways or controlling gene regulatory networks in organisms. However, chemical inducers must be added exogenously, leading to extra costs and potential barriers to scale-up. Expression systems that are induced by environmental changes avoid the need for costly chemical additions and allow bacteria to respond directly to growth conditions. The overall goal of this research is to develop small, synthetic, heat-repressible RNA thermosensors. These thermosensors are located in the 5' UTR upstream of the Shine-Dalgarno site, where they contain a cleavage site for ribonuclease E, an enzyme native to Escherichia coli that binds at the cleavage site and degrades the mRNA. At low temperatures, the recognition site is sequestered in a stem-loop structure. At high temperatures, the stem-loop unfolds and exposes the recognition site, and thus the mRNA is degraded, turning off gene expression. Synthetic heat-repressible RNA thermosensors were designed and tested in vivo by varying length, number of recognition sites, size of the loop, and predicted melting temperature of the stem. Cells containing a thermosensor-controlled GFP as well as an internal control RFP were grown at a variety of temperatures ranging from 15'C to 37'C, with the best performing thermosensor showing greater than 700-fold change in expression from low to high temperatures. These temperature-repressible thermosensors are small, do not require expression of protein regulators, and can function independently of expensive chemical inducers. In addition, they are simple to design and can be implemented in the optimization of a range of cellular processes.