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

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 used both stable and destabilized GFP, the latter with a half-life of eighty-one minutes in V. fischeri In culture, PhoB induced the reporter when phosphate was depleted below 10 μM. During symbiotic colonization of its host squid Euprymna scolopes , the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and re-engineering. IMPORTANCE Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between "on" and "off" states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over thirty-four billion sequence variants, we identified bases correlated with favorable performance by screening only eighty-three candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri This promoter-design strategy will facilitate the engineering of other regulator-specific reporters.