Engineered Stop\nand Go T7 RNA Polymerases

Precise, stringent, post-translational activation of\nenzymes is\nessential for many synthetic biology applications. For example, even\na few intracellular molecules of unregulated T7 RNA polymerase can\nresult in growth cessation in a bacterium. We sought to mimic the\nproperties of natural enzymes, where activity is regulated ubiquitously\nby endogenous metabolites. Here we demonstrate that full-length, single\nsubunit T7-derived RNA polymerases (T7 RNAP) can be activated by physiologically\nrelevant concentrations of indoles. We used rational design and directed\nevolution to identify T7 RNAP variants with minimal transcriptional\nactivity in the absence of indole, and a 29-fold increase in activity\nwith an EC<sub>50</sub> of 344 μM. Indoles control T7-dependent\ngene expression exogenously, endogenously, and between cells. We also\ndemonstrate indole-dependent bacteriophage viability and propagation\nin trans. Specificity of different indoles, T7 promoter specificities,\nand portability to different bacteria are shown. Our <u>l</u>igand <u>a</u>ctivated <u>R</u>NA <u>p</u>olymerases (LARPs) represent a new chemically inducible\n“stop and go” platform immediately deployable for novel\nsynthetic biology applications, including for modulation of synthetic\ncocultures.