De Novo Design of the ArsR Regulated P(ars) Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination

[Image: see text] Whole-cell biosensors for arsenic contamination are typically designed based on natural bacterial sensing systems, which are often limited by their poor performance for precisely tuning the genetic response to environmental stimuli. Promoter design remains one of the most important approaches to address such issues. Here, we use the arsenic-responsive ArsR-P(ars) regulation system from Escherichia coli MG1655 as the sensing element and coupled gfp or lacZ as the reporter gene to construct the genetic circuit for characterizing the refactored promoters. We first analyzed the ArsR binding site and a library of RNA polymerase binding sites to mine potential promoter sequences. A set of tightly regulated P(ars) promoters by ArsR was designed by placing the ArsR binding sites into the promoter’s core region, and a novel promoter with maximal repression efficiency and optimal fold change was obtained. The fluorescence sensor P(lacV)-P(arsOC2) constructed with the optimized P(arsOC2) promoter showed a fold change of up to 63.80-fold (with green fluorescence visible to the naked eye) at 9.38 ppb arsenic, and the limit of detection was as low as 0.24 ppb. Further, the optimized colorimetric sensor P(lacV)-P(arsOC2)-lacZ with a linear response between 0 and 5 ppb was used to perform colorimetric reactions in 24-well plates combined with a smartphone application for the quantification of the arsenic level in groundwater. This study offers a new approach to improve the performance of bacterial sensing promoters and will facilitate the on-site application of arsenic whole-cell biosensors.