De Novo Design of the ArsR Regulated P_ars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination

Abstract

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.

Natural Microbial Reactor-Based Sensing Platform for Highly Sensitive Detection of Inorganic Arsenic in Rice Grains

“…The current goals in the development of biosensors encompass achieving low background values, high sensitivity, high SNR, and a high output range. ,,− Promoter modifications play a crucial role in enhancing the output signal; however, stronger promoters can also lead to higher background expression, while weaker promoters result in lower output signals . In this study, a novel P ars promoter was designed by incorporating the ABS into the core region, resulting in significant improvement in repression efficiency and signal output levels . Additionally, the strategic placement of additional ABS elements upstream or downstream of the target promoter acted as transcription roadblocks, reducing background expression of downstream reporter genes.…”

“…44 In this study, a novel P ars promoter was designed by incorporating the ABS into the core region, resulting in significant improvement in repression efficiency and signal output levels. 46 Additionally, the strategic placement of additional ABS elements upstream or downstream of the target promoter acted as transcription roadblocks, reducing background expression of downstream reporter genes. This approach improved the SNR output, while maintaining the dose-dependent effect.…”

Synthetic bacteria for the detection and bioremediation of heavy metals

“…Consequently, the engineered P ars promoter was able to detect As as low as 10 nM. In a following study, the ArsR-binding site was placed to overlap the core region (between −35 and −10 site) of the P ars promoter after the alignment of ArsR-binding site sequence with a library of RNA polymerase binding sites (−35 and −10 sites) and −35 site restructure in ArsR-binding site sequence ( Chen et al, 2022 ). In addition, the ArsR expression level was also optimized.…”

Selection of Highly Specific DNA Aptamer for the Development of QCM‐Based Arsenic Sensor

“…[1] Arsenic groundwater contamination has been reported in over 70 countries. [2] There are various reports on arsenic detection, which comprise optical sensors, [3][4][5] electrochemical sensors, [6][7][8] Plasmonic sensors, [9,10] calorimetric sensors, etc. [11,12] Though different assays have achieved satisfying detection capabilities and selectivity, these techniques require labelling, multiple washing, and characterization steps.…”