Rapid prototyping of engineered transcription factor specificity using an in vitro transcription-translation (TX-TL) system

View all posters

Emmanuel Lorenzo de los Santos, Joseph T Meyerowitz, Richard M Murray

California Institute of Technology, United States

The ability to rapidly test and develop new components and subsystems for use in synthetic biology would broaden the repetoire from which new capabilities can be generated. Multiple techniques such as rapid DNA assembly, in vitro transcription-translation systems, and computational protein design may be combined to move from a notional function to proof-of-concept faster than would otherwise be possible. We show preliminary results from a new engineering process that combines computational protein design with a fast in-vitro “breadboarding” system for rapid, low-cost prototyping of a new biological transcription factor-based sensor. The multidrug response regulator qacR from S. aureus, which has been previously characterized, was chosen as a target for re-engineering. Computational protein design targeting the qacR transcription factor’s small molecule binding site was performed, resulting in a set of mutants designed to switch the specificity of qacR to a new small molecule ligand. These mutants are characterized using an in vitro transcription-translation (TX-TL) system. In this process, the TX-TL system provides multiple advantages over the characterization of these transcription factors in vivo. The protein is being engineered to detect a particularly toxic small molecule found in lignocellulosic feedstock. TX-TL allows us to screen for transcription factor sensitivities above concentrations that can be realized inside cells (either due to toxicity or transport), making it possible to characterize potential hits for activities otherwise inaccessible via in vivo screening. Furthermore, control of expression levels of different system components can be achieved by varying the DNA concentrations in the in vitro system, avoiding time-consuming fine tuning of plasmid copy number and promoter strength in early system design phases. As a whole, our new engineering process provides quantitative information about the circuit within hours of setup over a variety of different conditions, enabling cost-effective and rapid protoyping of experimental designs.