Massively Parallel Double-Selection Workflow for the Evolution of Molecular Switches Based on Surface-Display in Escherichia coli.

Microbial surface display of proteins is a versatile method for a wide range of biotechnological applications. Herein, the use of a surface display system in E. coli for the evolution of a riboswitch from an RNA aptamer is presented. To this end, a streptavidin-binding peptide (SBP) is displayed at the bacterial surface, which can be used for massively parallel selection using a magnetic separation system. Coupling gene expression from a riboswitch library to the display of SBP hence allows selection of library members that express strongly in the presence of a ligand. As excessive SBP expression leads to bacterial growth inhibition, it can be used to negatively select against leaky riboswitches expressing in the absence of ligand. Based on this principle, we devise a double selection workflow that enables quick selection of functional riboswitches with a comparatively low screening workload. The efficiency of our protocol by re-discovering a previously isolated theophylline riboswitch from a library was demonstrated, as well as a new riboswitch that is similar in performance, but slightly more responsive at low theophylline concentrations. Our workflow is massively parallel and can be applied to the screening or pre-screening of large molecular libraries.