Optimization of a Cell-Free Protein Expression System for Improved Functionality of Natural Transcriptional Riboswitches

Riboswitches (RS) are naturally occurring RNA regulatory elements capable of controlling gene expression by inducing structural changes in the 5’ untranslated regions of mRNA following binding of a specific analyte. We are particularly interested in leveraging the ability of riboswitches to recognize specific ligands to develop biosensors with more accurate and sensitive detection for biomarkers of interest. While there has been much effort to standardize and optimize cell-free protein expression system (CFPS) reactions for maximal protein expression with T7 and native bacterial promoters, these systems typically perform poorly when used to study RS functionality, exhibiting a lack of switching and significant background expression in the OFF state. Here we optimized a CFPS to experimentally identify the critical conditions required for functionality of two naturally occurring RS from Bacillus subtilis - the pbuE RS and the flavin mononucleotide (FMN) RS. The pbuE RS is a transcriptional ON switch, upregulating expression of downstream genes in response to increased concentrations of adenine or the 2-aminopurine analog, while the FMN RS is a transcriptional OFF switch, downregulating expression of downstream genes in the presence of FMN. Cellular extract from the E. coli K12 parental strain BW25113 was optimized to maximize the activation ratio of each RS under control of a native bacterial promoter and upstream of a fluorescent reporter protein. Extract preparation and reaction setup were adapted from a protocol optimized for maximal protein production from a native bacterial promoter in BL21star(De3) extract. We identified key aspects of extract preparation required for functionality of these RS that led to increased fluorescent signal and increased activation ratio in the presence of analyte. Future studies will apply these optimizations to additional riboswitches, both natural and synthetic. A better understanding of transcriptional RS functionality in CFPS will further the utility of these RNA sensing elements as biosensors.