Bacterial transcription is regulated by intracellular and extracellular environmental signals that allow cells to respond to their dynamic environment. These transcriptional regulators have been used in biotechnology for coordinating protein expression, for dynamic regulation of metabolic pathways, and for engineering cellular phenotypes. Whole cell biosensors can be created to report on the environmental signal that controls transcriptional regulation when fluorescent proteins are placed downstream of cognate promoters. The current limitation of such technologies is the limited set of characterized and specific transcriptional regulators. In order to address this need, we are developing new transcriptional regulators using phage assisted continuous directed evolution. The method involves the continuous evolution of a phage encoding transcriptional regulator genes containing advantageous mutations. Favorable mutations are selected by the rate at which phage are reproduced. 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. We will describe our results showing phage production linked to a novel transcriptional regulatory activity. We will describe the important factors for continuous evolution and described our efforts to optimize the system for efficient selection.

Toward engineering bacterial transcriptional regulators through continuous directed evolution

249th National Meeting (2015)

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