A Conditional Degron Tag for RNA Sensing in Bacteria

Abstract

Here we report the development of a conditional degron system for use in bacteria in which a tagged fluorescent protein (FP) reporter, mVenus-S1, is stabilized only in the presence of TAR RNA. The functional design takes advantage of the bipartite nature of the ssrA degron sequence, so that TAR RNA binding to the inserted Tat peptide sequence blocks degron recognition to rescue protein activity. Several fluorescent proteins and a chemiluminescent enzyme can be tagged and activated using this system, and our results reveal that chromophore maturation time correlates with fold change, with up to [~]60-fold turn-on. Finally, we designed a functional biosensor for oxyS sRNA and integrated it into a single transcript in which the FP and RNA-based biosensor are encoded on the same mRNA. To our knowledge, this is the first proof-of-concept for a novel biosensor design that expresses both protein and RNA components from a single transcript. The streamlined system is fully genetically encoded, does not require an exogenous fluorophore, and permits both components to be coordinated by expression from the same promoter.