Screening Synthetic RNA Thermometers in Escherichia coli: A Single-Marker Dual-Selection Approach Using TetA

Natural regulatory RNAs, such as riboswitches and RNA thermometers (RNAT), hold significant promise within the field of synthetic biology. A variety of methodologies have been established to control inducible gene expression; however, these approaches typically involve multi-component systems that include cis-acting elements at the DNA or RNA level, as well as trans-acting regulatory proteins and small molecules that serve as inducers. RNA thermometers constitute one of the rare instances of single-component regulators of gene expression. They feature a temperature-sensitive secondary structure situated in the 5’ untranslated region of mRNA, which includes the ribosome-binding site. An increase in temperature leads to a gradual transition in the equilibrium between the closed and open forms, promoting the open conformation in a zipper-like manner, thus improving the efficiency of translation initiation. RNATs have the potential to be used independently of the organism in which they are placed, based on the secondary structure alone. Moreover, regulation through temperature control can reduce industrial production costs and avoid the possible toxicity of inducers. Although the production of large synthetic RNAT libraries is feasible, there is a bottleneck in selecting and screening positive candidates. To tackle this issue, I propose the development of a positive and negative selection platform based on the TetA efflux pump as a single selection marker. The employment of tetracycline and nickel enables the selection of variants that are either open or closed at the desired temperatures. To validate the efficacy of the platform, an RNA thermometer library was analysed, leading to the identification of two novel synthetic RNATs exhibiting a fold change of 13.8-fold and 29.5-fold.