Sort-seq approach to engineering an E. coli formaldehyde-inducible promoter

Conference Dates

July 16-20, 2017


Tight and tunable control of gene expression is a highly-desirable goal in synthetic biology and metabolic engineering for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence-function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence-function relationships in a robust and rapid way.1, 2 Here we utilize a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) at a single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was generated and cloned upstream of a plasmid-borne gfp reporter. The library was partitioned into bins via FACS based on GFP expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. 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.

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