(335e) Sort-Seq Approach to Engineering an E. coli Formaldehyde-Inducible Promoter

Complex synthetic regulatory networks require orthogonal transcription factors with preferably modular DNA-binding and sensing domains. When controlling the expression of measurable reporters, such as fluorescent proteins or antibiotic resistance markers, these transcription factor-based biosensors have shown promise by increasing the efficiency of high-throughput screens and selections, and in dynamic pathway regulation by allowing real-time monitoring of intracellular metabolite concentrations. Here we expand the synthetic biology toolbox through the characterization of an E. coli transcription factor-based formaldehyde biosensor and engineer its corresponding operator sequence guided by information obtained through a robust and rapid sort-seq approach to achieve tight and tunable gene expression. The resulting analysis identifies two 19-bp repressor binding sites, one upstream of the -35 RNA polymerase (RNAP) binding site and one overlapping with the -10 site, and assesses the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range.