Optimization of Transcription Factor and Bacterial Promoter-Based Biosensors in Cell-Free Systems for Defense Applications

Cell-free biosensors have the potential to address a variety of sensing applications for the US Air Force and Department of Defense due to their high sensitivity and specificity for performance biomarkers and analytes of interest. Additionally, the ability to immobilize cell-free protein expression systems on paper allows for the establishment of ruggedized sensors for field applications. Here, we demonstrate the adaptation of several transcription factor-based biosensors to a cell-free platform optimized for expression of bacterial promoters. A cortisol sensor dependent on a LysR-family transcription factor was tested both in vivo with E. coli Nissle 1917 and in vitro with an E. coli BL21* DE3-derived cell-free system. Interestingly, the sensor exhibited much higher sensitivity in the cell-free reactions than in vivo, suggesting additional rate limiting events in the cell. Furthermore, we were able to successfully transfer the cortisol sensor to a paper-based cell-free platform. We also demonstrated the functionality of a deoxycholate sensor dependent on BreR, a TetR-like transcription factor and repressor from Vibrio cholorae, in our BL21* DE3 system. The sensor responded to deoxycholate in a dose-dependent manner in the cell-free system, but lacked reproducibility when the BreR and GFP reporter proteins were expressed simultaneously in the same reaction. Generation of extract pre-enriched with the BreR transcription factor provided a more robust and reproducible sensing platform. In addition, we have shown the functionality of a mercury (II) sensor dependent on MerR, a repressor/activator transcription factor from Shigella flexneri, in a bacterial RNA polymerase-dependent cell-free system. Combined our data shows the versatility of cell-free systems for the development of transcription-factor based sensors derived from a variety of bacterial species. Ongoing work includes the optimization of these sensors for increased sensitivity to detect biologically relevant concentrations of analytes and adaptation of these sensors for use on paper-based systems for field applications.