Using the promoters of MerR family proteins as “rheostats” to engineer whole-cell heavy metal biosensors with adjustable sensitivity

et al. 2021

Front. Microbiol.

Cadmium (Cd) is carcinogenic to humans and can accumulate in the liver, kidneys, and bones. There is widespread presence of cadmium in the environment as a consequence of anthropogenic activities. It is important to detect cadmium in the environment to prevent further exposure to humans. Previous whole-cell biosensor designs were focused on single-sensing constructs but have had difficulty in distinguishing cadmium from other metal ions such as lead (Pb) and mercury (Hg). We developed a dual-sensing bacterial bioreporter system to detect bioavailable cadmium by employing CadC and CadR as separate metal sensory elements and eGFP and mCherry as fluorescent reporters in one genetic construct. The capability of this dual-sensing biosensor was proved to simultaneously detect bioavailable cadmium and its toxic effects using two sets of sensing systems while still maintaining similar specificity and sensitivity of respective signal-sensing biosensors. The productions of double-color fluorescence were directly proportional to the exposure concentration of cadmium, thereby serving as an effective quantitative biosensor to detect bioavailable cadmium. This novel dual-sensing biosensor was then validated to respond to Cd(II) spiked in environmental water samples. This is the first report of the development of a novel dual-sensing, whole-cell biosensor for simultaneous detection of bioavailable cadmium. The application of two biosensing modules provides versatile biosensing signals and improved performance that can make a significant impact on monitoring high concentration of bioavailable Cd(II) in environmental water to reduce human exposure to the harmful effects of cadmium.

Section: Resultsmentioning

confidence: 99%

Detection of Bioavailable Cadmium by Double-Color Fluorescence Based on a Dual-Sensing Bioreporter System

Hui¹,

et al. 2021

Front. Microbiol.

“…The maximum binding capacity of recombinant cells with surface-exposed MerR under the control of a strong lac promoter was around 120 μmol/g cell [ 11 ]. Although the strength of the natural mer promoter is significantly weaker than the strength of the commercial inducible promoter, rational genetic designs have been proven to improve the sensitivity of the mer promoter [ 29 , 44 ]. In order to improve the Hg(II) binding capacity, the optimized genetic elements will be used to assemble an artificial mer operon to improve the amount of surface displayed Hg(II)-binding protein with low concentrations of Hg(II) exposure in our future studies.…”

Section: Resultsmentioning

confidence: 99%

Versatile artificial mer operons in Escherichia coli towards whole cell biosensing and adsorption of mercury

Zhang

et al. 2021

PLoS ONE

Mercury exists naturally and mainly as a man-made pollutant in the environment, where it exerts adverse effects on local ecosystems and living organisms. It is important to develop an appropriate synthetic biological device that recognizes, detects and removes the bioavailable fraction of environmental mercury. Both single-signal and double-signal output mercury biosensors were assembled using a natural mer operon as a template. Selectivity and sensitivity of whole-cell biosensors based on artificial mer operons were determined. Three whole-cell biosensors were highly stable at very high concentrations of mercuric chloride, and could detect bioavailable Hg(II) in the concentration range of 6.25–200 μM HgCl2. A novel Hg(II) bioadsorption coupled with biosensing artificial mer operon was assembled. This would allow Hg(II)-induced Hg(II) binding protein cell surface display and green fluorescence emission to be achieved simultaneously while retaining the linear relationship between fluorescent signal and Hg(II) exposure concentration. The present study provides an innovative way to simultaneously detect, quantify, and remove bioavailable heavy metal ions using an artificially reconstructed heavy metal resistance operon.

“…Rational design of genetic circuit is another key factor for improved performance of biosensors except for the optimization of detection conditions. The expression level of sensor MerR was used as a regulator in the genetic circuit for tuning detection sensitivity, and the detection limit of exponential-phase culture of biosensor with GFP as an output signal was enhanced to 0.1 μM Hg(II) 45 . A novel Hg(II) biosensor, which could detect concentrations as low as 0.02 μM Hg(II), was developed by combining quorum sensing-based positive feedback systems.…”

Section: Discussionmentioning

confidence: 99%

Development of a bioavailable Hg(II) sensing system based on MerR-regulated visual pigment biosynthesis

Hui²,

Liu

et al. 2021

Sci Rep

Engineered microorganisms have proven to be a highly effective and robust tool to specifically detect heavy metals in the environment. In this study, a highly specific pigment-based whole-cell biosensor has been investigated for the detection of bioavailable Hg(II) based on an artificial heavy metal resistance operon. The basic working principle of biosensors is based on the violacein biosynthesis under the control of mercury resistance (mer) promoter and mercury resistance regulator (MerR). Engineered biosensor cells have been demonstrated to selectively respond to Hg(II), and the specific response was not influenced by interfering metal ions. The response of violacein could be recognized by the naked eye, and the time required for the maximum response of violacein (5 h) was less than that of enhanced green fluorescence protein (eGFP) (8 h) in the single-signal output constructs. The response of violacein was almost unaffected by the eGFP in a double-promoter controlled dual-signals output construct. However, the response strength of eGFP was significantly decreased in this genetic construct. Exponentially growing violacein-based biosensor detected concentrations as low as 0.39 μM Hg(II) in a colorimetric method, and the linear relationship was observed in the concentration range of 0.78–12.5 μM. Non-growing biosensor cells responded to concentrations as low as 0.006 μM Hg(II) in a colorimetric method and in a Hg(II) containing plate sensitive assay, and the linear relationship was demonstrated in a very narrow concentration range. The developed biosensor was finally validated for the detection of spiked bioavailable Hg(II) in environmental water samples.