(188f) Designing and Screening Protein-Sensing Riboswitches in an All-E. coli Cell-Free Expression System

Proteins are important biomarkers of human disease, indicating the presence of medical conditions like cardiovascular disease and different cancers. Here, we develop a screening platform for translationally-acting riboswitches that can detect proteins, including biomarkers of human disease, using an inexpensive TX-TL cell-free expression system. Using a statistical thermodynamic model of translational riboswitch regulation, we designed riboswitches that bind to and detect the viral coat protein MS2, as well as the biomarkers C-reactive protein and interleukin 32. By coexpressing the riboswitch and the protein which it detects in TX-TL, we observed up to 14-fold activation in the production of mRFP1. To further examine how translational regulation of gene expression differs between in vivo and in vitro environments, we observed how the expression of a library of ribosome binding sites of different strengths differed in TX-TL. As compared to expression in vivo, the dynamic range of translation decreased. We further manipulated the in vitro environment by introducing the synthetic crowding agents PEG-8000 and Ficoll 400 to better mimic the in vivo environment. These synthetic crowders increased the absolute levels of gene expression for all constructs tested, but further decreased the dynamic range of gene expression in TX-TL.