Sort-seq approach to engineering an E. coli formaldehyde-inducible promoter
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.
Julia Rohlhill, Eleftherios T. Papoutsakis, and Nicholas R. Sandoval, "Sort-seq approach to engineering an E. coli formaldehyde-inducible promoter" in "Biochemical and Molecular Engineering XX", Wilfred Chen, University of Delaware, USA Nicole Borth, Universität für Bodenkultur, Vienna, Austria Stefanos Grammatikos, UCB Pharma, Belgium Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/biochem_xx/9