Development of a Highly Sensitive Whole-Cell Biosensor for Arsenite Detection through Engineered Promoter Modifications

Abstract

Whole-cell biosensors have attracted considerable interests because they are robust, eco-friendly, and cost-effective. However, most of the biosensors harness the naturally occurring wild-type promoter, which often suffers from high background noise and low sensitivity. In this study, we demonstrate how to design the core elements (i.e., RNA polymerase binding site and transcription factor binding site) of the promoters to obtain a significant gain in the signal-to-noise output ratio of the whole-cell biosensor circuits. As a proof of concept, we modified the arsenite-regulated promoter from Escherichia coli K-12 genome, such that it has a lower background and higher expression. This was achieved by balancing the relationship between the number of ArsR binding sites (ABS) and the activity of the promoter and adjusting the location of the auxiliary ABS. A promoter variant ParsD-ABS-8 was obtained with an induction ratio of 179 (11-fold increase over the wild-type promoter) when induced with 1 μM arsenite. Importantly, the developed biosensor exhibited good dose–response in the range of 0.1 to 4 μM (R 2 = 0.9928) of arsenite with a detection limit of ca. 10 nM. These results indicated that the engineered promoter modification approach could be used to improve the performance of whole-cell biosensors, thereby facilitating their practical application.

Long-Term Rewritable Report and Recording of Environmental Stimuli in Engineered Bacterial Populations

“…Arsenic is a common heavy metal pollutant, which occurs naturally in the earth’s crust and is widely distributed in air, water, and even food, posing a serious threat to human health, such as causing neurological diseases, renal system diseases, cardiovascular diseases, and skin lesions and increasing the risk of liver, bladder, lung, and skin cancers simultaneously. In the past decade, whole-cell biosensors have been developed for the detection of arsenic contamination. , However, no biosensor has been reported that can rapid report and long-term record the presence of arsenite. Based on our previous work, we constructed an arsenite-responsive plasmid called pWT-arsR (Figure A).…”

“…In the past decade, whole-cell biosensors have been developed for the detection of arsenic contamination. 34,35 However, no biosensor has been reported that can rapid report and long-term record the presence of arsenite. Based on our previous work, 35 we constructed an arsenite-responsive plasmid called pWT-arsR (Figure 3A).…”

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

“… 37 Its product is a green fluorescent protein with a long half-life. The arsR gene with the constitutive promoter P J109 and the ribosome binding site (RBS) B0030 was amplified from the J109-ParsD-ABS-2 plasmid 12 and inserted into the Hin dIII and Sac I sites of the sensor detection platform pPROBE-TT plasmid using a recombination reaction to generate the p-TT-P J109 -r30 arsR plasmid ( Figure S1 ). Primer extension polymerase chain reaction (PCR) was used to obtain the wild-type P ars promoter and variant promoters with a refactored −35 site at varying locations.…”

“…To change the density of the receptor protein ArsR, the P lacV promoter was used to drive ArsR expression. The P arsXX gene with the mutation was amplified from the p-TT-P J109 -r30 arsR -P arsXX plasmid by PCR and then ligated with Eco RI and Sac I restriction-enzyme-digested p-TT-lacV arsR 12 to generate p-TT-P lacV -arsR- P arsXX plasmids, which were named P lacV -P arsXX . For the construction of the sensor with lacZ as the reporter, lacZ with the same RBS as gfp was PCR-amplified from the wild-type E. coli MG1655 genome and then ligated with Eco RI and Hap I restriction-enzyme-digested P lacV -P arsXX to generate P lacV -P arsXX - lacZ .…”

Development and optimization of N‐acetylneuraminic acid biosensors in Bacillus subtilis

“…Although we obtained a NeuAc biosensor with a relatively low level of expression leakage, further efforts are required to reduce the expression leakage levels and obtain a significant improvement in the signal-to-noise output ratio. Further optimization of the RNA polymerase binding site and the transcription factor binding site through promoter engineering are required to improve the biosensor [22]. To more accurately determine the characteristics of the NeuAc biosensor, we added NeuAc to the medium at 0 to 5.0 g/L and continuously measured GFP expression for 42 H. The biosensor was shown to have a two-step activation, firstly at 0-0.1 g/L NeuAc and then at 2-3 g/L NeuAc.…”