(228di) Using Phage-Assisted Continuous Evolution to Develop Novel Biosensors

Transcriptional regulators are proteins that enable cells to dynamically alter their gene expression in response to environmental cues. The modularity of the sensor-regulator design of transcription factors has been frequently used in biotechnology for control over protein expression in engineered phenotypes. Complex products require multistep pathways; however, balancing these pathways proves difficult due to the limited set of characterized and specific transcriptional regulators. There is a need in the field to develop transcriptional regulators that respond to novel signals. This would address the lack of regulators available as well as enable development of whole-cell biosensors for detection of molecules external to cells. Directed evolution is a commonly used technique to rationally design novel proteins, but requires a priori knowledge that may not always be available. Alternatively, phage-assisted continuous evolution (PACE) is a method involving the continuous propagation a phage encoding transcriptional regulator genes harboring advantageous mutations. Selection pressure is driven by phage replication rate. The critical selection occurs when the new transcriptional regulatory activity results in the production of the pIII phage coat protein, which is responsible for phage infectivity. Without proper production of pIII, mutations are not propagated and the phage population is instead saturated with mutants able to successfully produce pIII. PACE allows for rapid, autonomous evolution of protein-ligand interactions without a priori knowledge. We will describe our results showing phage production linked to a novel transcriptional regulatory activity.