Generating proteins with radically altered activities and functions is a major goal in synthetic biology. While in vivo directed evolution is generally capable of achieving this goal, it requires evolutionary stepping stones that enable a slowly progressing adaptation of the evolving gene pool to the novel, desired function. To implement such selection pressure modulation, a set of fine-tuned accessory plasmids has been required in the past. These then need to be applied in a well-orchestrated fashion to slowly increase selection pressure over time, facilitating genetic drift and eventually emergence of the novel function.
Here, we present OptoSELECT, an optogenetic selection stringency modulator able to easily tune selection pressure during PREDCEL and PACE experiments using defined doses of blue light. Our tool is based on the previously described, blue light-dependent transcription factor EL222 and a bidirectional promoter system, which either induces or represses the expression of M13 geneIII upon blue light irradiation. Using OptoSELECT, we demonstrate successful light control of geneIII-dependent phage propagation using an engineered pBLind promoter-geneIII expression cassette. Our work paves the way towards simple and straight-forward optimization of in vivo evolution experiments by optogenetic modulation of the selection pressure.
