Can the Development of a Dual-Responsive Whole-Cell Biosensor be Used to Detect Mercury and Arsenic in Drinking Water?

Abstract:

Millions of people have no access to running water, and instead rely on groundwater to drink. Yet, groundwater often contains dangerous pollutants, and heavy metals like arsenic and mercury cause health issues worldwide. Traditional methods to detect metals in water are expensive and inaccessible. This study presents a dual-responsive whole-cell biosensor engineered in Escherichia coli, designed to detect the presence of Hg2+ and As3+ through distinct genetic circuits that trigger green and red fluorescence. Utilizing MerR and ArsR metal-sensing regulators, the whole-cell biosensors can visually indicate the presence of mercury and arsenic. To evaluate potential in field-deployment, the biosensor is sealed in a capsule, which can function as a simple testing kit to detect mercury and arsenic.

 

Introduction:

Access to clean water is a fundamental necessity for human health; yet, the pervasive abundance

of groundwater pollution remains an alarming global crisis. Heavy metal pollutants, like mercury and arsenic, are among the most toxic. Virtually undetectable without lab equipment, metals like arsenic are found in over 50% of groundwater samples in Africa, and when 115 million people get their water from untreated groundwater sources, heavy metal pollution cannot be understated (Irunde et al., 2022). Despite the chronic health problems and cancer risk associated with metals, ways to test groundwater for metals are not feasible in third-world countries. However, whole-cell biosensors hold potential for fast and cheap metal detection. Using genetic circuits, biosensors activate a visual indicator (like fluorescence) when certain metallic ions are detected. Single-metal biosensors exist, and have been proven to be successful; yet due to the wide variety of heavy metals in water, they have not been applied to metal detection in drinking water. This study aims to develop a dual-responsive biosensor using Escherichia coli engineered with distinct genetic circuits to detect mercury and arsenic ions via green and red fluorescence, respectively. With the cheap production of these biosensors, they could be encapsulated following lyophilization (freeze-drying), and distributed across struggling villages to quickly and affordably test their drinking water.

 

Literature Review:

Current methods to detect the presence of heavy metals are expensive, slow and

inaccessible (Petrusevski, Sharma, Schippers, & Shordt, 2007). However, whole-cell biosensors show promise. Zevallos-Aliaga et al. (2024) engineered a biosensor that successfully exhibited luminescence in the presence of mercury ions. Utilizing genetic circuit principles, they encoded a MerR regulator with a Pmer promoter upstream to a reporter gene, and demonstrated the biosensor’s ability to detect mercury in water. Similarly, Liu et al. (2022) synthesized a new genetic circuit with the Pars promoter, effectively redesigning it to enhance the sensitivity of the biosensor, lowering detection levels down to 3.75 parts per billion—ten times less than previously established, and under the WHO safety limit of 10 ppb (World Health Organization, 2018). Zhang et al. (2021) coupled the concepts present in traditional genetic circuits to create a dual-sensing system. While Zhang used two separate regulators for cadmium, his research proved the viability of an avenue to create a dual-sensing biosensor with two separate metals. Using the existing breakthroughs of Liu and Zhang alongside Zevallos-Aliaga’s foundation of a metal-inducible gene expression system, the success of a dual-sensing biosensor of mercury and arsenic can be hypothesized.

 

Methodology:​ 

Genetic Circuit Design: Initially, the in silico (virtual) construction of two discrete, metal-inducible gene

expression circuits will have to be developed. To create a whole-cell biosensor for both mercury and arsenic, separate metal-responsive genetic circuits will have to be synthesized.The synthetic operon developed in the mercury responsive circuit will comprise of the Pmer promoter, regulated by an MerR transcriptional activator located upstream of the gfpmut3a reporter gene encoding green fluorescent protein (GFP). In the presence of mercury ions (Hg2+),, MerR binds with Hg2+ and undergoes an allosteric conformational shift; the protein morphs its conformation, allowing for transcriptional activation at Pmer and resulting in the upregulation of GFP expression (Nascimento & Chartone-Souza, 2003; Virta et al., 1995). An arsenic responsive circuit will be constructed in a similar fashion. However, this second operon will utilize the Pars promoter, and be regulated by the ArsR repressor, which will control transcription of the rfp gene. ArsR dissociates from the Pars operator site in the presence of arsenic ions (As3+), derepressing transcription and enabling RFP expression—a key difference from the MerR activator (Chen et al., 2022). Both circuits will be designed with a high-copy plasmid vector codon-optimized for E. Coli and a unique antibiotic selection marker (e.g. pSB1C3 with AmpR and KanR) (Che, 2008; Jenkins et al., 2023). To avoid cross-talk between the dual-circuit system, non overlapping promoter regions will be embedded into the circuit, essential for subsequent combination into one chassis (Zevallos-Aliaga et al., 2024). Utilizing public bioinformatics softwares and databases (e.g. Benchling and Addgene), constructs will be designed and submitted to a gene synthesis company for production. Transformation and Culture: A non-pathogenic strain of E. Coli will be used as the chassis of choice. Using calcium chloride (CaCl2), the chassis will be made chemically competent. Both plasmids will be inserted into the traditional chassis via heat shock transformation and an ice bath, outlined in Chang et al., 2017. The resulting transformed cells will be cultured on traditional LB Agar plates, containing ampicillin and kanamycin, as to cull cells lacking both genetic circuits containing selection markers AmpR and KanR, respectively. This cultivation will ensure solely transformed bacteria with a dual-circuit system will be selected. Fluorescence Response Testing and Capsule Simulation: Transformed E. Coli cells will be exposed to varying concentrations of Hg2+ and As3+ to assess biosensor responsiveness. The output signal—green and red fluorescence—will be visually measured through the exposure to UV light. Testing will be done in triplicate with positive and negative controls. To simulate field use, transformed cells will be lyophilized (freeze-dried) with a cryoprotectant (e.g trehalose), and sealed into a small capsule (Farr 2021). Lyophilized cells will be resuscitated through hydration with water. Through capsule storage, the feasibility of rehydration, glow strength in low-resource conditions, and stability over long-term storage will be evaluated. Data Analysis: Results will be analyzed for sensitivity (lowest concentration for metals to induce fluorescence), specificity (response variances between Hg2+,  As3+ , and non-target metals), response time, and relative signal brightness. Findings will provide insight on the feasibility and field-readiness of the whole-cell biosensor.

 

Conclusion:

This study developed a dual-responsive biosensor in E. coli using both MerR-Pmer-GFP and ArsR-Pars-FRP systems. If successful, this biosensor could offer an accessible solution to testing water qualities in third-world countries facing pollution. While long-term problems with storage may be present, the principle of this design can be applied and broadened to encapsulate many metals, creating a single tool to test water quality.

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