Programmable allosteric transcription factors as specific molecular sensor-actuators

Engineering biology through design-build-test cycles is most often limited by the test phase, due to low-throughput phenotype evaluation. This is especially true for the production of high-value target molecules. This application will establish a platform to reprogram natural allosteric transcription factors to specifically recognize new target small molecules, resulting in intrinsically coupled sensor-actuators programmed to a user-defined target. The platform includes three main stages, followed by specificity tuning: 1) computationally predict a large number of sensor candidate designs for response to a target molecule; 2) assemble large libraries of predicted sensor genes from microarray-synthesized DNA; and 3) evaluate the designs for new ligand binding performance using a three-tiered, high-throughput screen. We then mature the response of the candidate sensors for higher activation and to reduce promiscuity for the original effector. Natural allosteric transcription factors encompass a large range of effector molecules, so this strategy is broadly extensible to many target molecule classes. Successful completion of this application provides a robust platform to reprogram allosteric transcription factors to serve as sensor-actuators for user-defined applications.