An iterative synthetic approach to engineer a high-performing PhoB-specific reporter.

An iterative synthetic approach to engineer a high-performing PhoB-specific reporter. Appl Environ Microbiol. 2018 May 11;: Authors: Stoudenmire JL, Essock-Burns T, Weathers EN, Solaimanpour S, Mrázek J, Stabb EV Abstract Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal "on" expression when PhoB is active, minimal background in the "off" state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at nineteen additional intervening nucleotide positions for which we did not predict sequence-specific effects the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri, enabling us to identify bases at particular randomized positions that significantly correlated with high "on" or low "off" expression. A second round of promoter design rationally constrained thirteen additional positions, leading to a reporter with high PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we us...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research