Introduction

Detecting inorganic and organic mercury typically requires complex instruments, sophisticated facilities, and highly trained personnel. However, alternatives such as microbial biosensors have proven to be practical and effective tools for mercury detection (Dennison and Turner, 1995). Organic mercury species, particularly methylmercury, are highly toxic and are primarily ingested through food (Tang et al., 2020). Therefore, it is crucial to develop methods that enable the selective detection of organic mercury. The discovery of bacterial mercury detoxification mechanisms has made it possible to leverage these systems—which localize and volatilize both inorganic and organic mercury—for detection. In 2001, the first biosensor for organic mercury detection was developed (Han et al., 2001).

This advance was enabled by the use of the MerB enzyme and the MerR transcription factor, which regulate the expression of a measurable reporter. However, the initial biosensor was unable to differentiate between inorganic mercury and cadmium. Advances in synthetic biology and directed evolution have now provided new proteins and strategies that allow for the specific detection of the major forms of mercury contamination in food (Hui et al., 2024). Our goal is to develop a portable, sensitive, and selective microbial biosensor specifically for the detection of methylmercury. To achieve this, we propose two main strategies to tackle the challenge.

The Mercury Biosensor: Engineering Nature for Pollution Detection

The iGEM Bolivia team has developed a biosensor that detects both organic and inorganic mercury using engineered E. coli DH5α. The system is built around two distinct strategies to ensure high sensitivity and selectivity. Let's explore these strategies in detail:

Chassis: E. coli DH5α

We selected E. coli DH5α as the host strain due to its efficient uptake of constructs and its ability to maintain stable modified DNA. This strain is non-toxic to mercury and is easy to handle in standard lab conditions. The rapid reproduction rate of DH5α also supports quick bacterial growth, making it a scalable platform for biosensor development (Kostylev et al., 2015).

Design Objective

Our objective is to create a portable, sensitive, and selective microbial biosensor that distinguishes between organic mercury (methylmercury) and inorganic mercury (Hg²⁺). This system is critical for field monitoring of mercury pollution due to the high toxicity of methylmercury (Tang et al., 2020).

Construct Overview and Their Roles

The biosensor utilizes three unique construct, each designed to detect and detoxify mercury with high selectivity and efficiency:

• Construct 1: Equipped with merRm4-1, which is sensitive to both organic and inorganic mercury (Zhu et al. 2023). When methylmercury is detected, it activates mRFP_Magenta, producing magenta fluorescence. TetR blocks this signal in the presence of inorganic mercury (Hg²⁺).
• Construct 2: Contains merRtn501 (Lian et al., 2014), which detects inorganic mercury (Hg²⁺) and triggers the expression of mChartreuse, producing green fluorescence. TetR is also expressed, binding to the tetO site to prevent magenta fluorescence when Hg²⁺ is detected.
• Construct 3: Contains MerA, gomerB, and gdh, forming the detoxification system. MerA converts toxic Hg²⁺ into harmless gaseous Hg⁰. gomerB breaks down methylmercury into Hg²⁺ (Guo et al., 2024), which MerA can then detoxify. gdh provides NADPH, powering this reduction process.

Strategy 1: The "NOT Logic Gate" for Organic Mercury Detection

This strategy is designed to selectively detect organic mercury while preventing false signals from inorganic mercury. It utilizes a dual-construct system that ensures precision in detection Dual-Plasmid System: The bacterium is transformed with Plasmids #1 and #2.

This system provides selective signaling, with magenta fluorescence for organic mercury and green fluorescence for inorganic mercury, while TetR prevents overlap between the signals.

Strategy 2: Enzymatic Volatilization of Inorganic Mercury Using the DogCatcher-DogTag System

This strategy eliminates inorganic mercury (Hg²⁺) by converting it into non-toxic elemental mercury (Hg⁰), which is volatile and can be safely released. It uses a dual-plasmid approach, combining Plasmid 2 and Plasmid 3, with the DogCatcher-DogTag system for enzyme localization at the bacterial membrane.

Mechanisms for Organic Mercury Selectivity:

1. Selective Entry of Methylmercury:

   Contains merRm4-1, a transcription factor sensitive to both organic and inorganic mercury. When organic mercury (methylmercury) binds to merRm4-1, it activates mRFP_Magenta, resulting in magenta fluorescence, indicating organic mercury’s presence.

2. Surface Display Systems with DogCatcher-DogTag:

   Mercury salts can enter the cell via water-soluble protein transporters in the bacterial membrane. To prevent this, we developed a system to anchor the mercury reductase enzyme to the outer membrane, where Hg²⁺ ions are attracted and reduced to Hg⁰. While elemental mercury can enter the cell, it does not activate the reporter gene. The transporter used to localize proteins to the membrane is Lpp-OmpT, which anchors the DogCatcher proteins. DogTags, which bind to DogCatchers, transport proteins to the outer membrane. Gene fusions Lpp-OmpT-DogCatcher and MerA-DogTag were included on the same plasmid.
   Mercury reductase (MerA), an intracellular enzyme requiring cofactors, is anchored to the outer membrane. To support this, we incorporated a cofactor regeneration system. MerA requires NADPH, which is replenished by glucose dehydrogenase (GDH). GDH uses glucose as its substrate, ensuring a constant supply of NADPH for the detoxification process. Both GDH and MerA genes are fused with DogCatcher for membrane localization.

3. Detection of methylmercury

   To detect methylmercury, the system uses the alkylmercury lyase gomerB, which reduces methylmercury to Hg²⁺. This inorganic mercury binds to the MerR protein on a second plasmid, triggering the expression of a green fluorescent protein (GFP), indicating the presence of mercury.

4. Controlled proteolysis

   To prevent MerA from prematurely reducing Hg²⁺ (converted by gomerB), a controlled proteolysis system was incorporated. This system includes a TEV protease on the second plasmid that degrades MerA when it encounters Hg²⁺ in the cytoplasm. When MerA is located on the outer membrane, the TEV protease cannot degrade it, ensuring proper detection of organic mercury.

   • Construct 2: Detection of Inorganic Mercury

     Construct 2 detects inorganic mercury (Hg²⁺) using the merRtn501 gene, which encodes a transcription factor that binds to Hg²⁺. This binding activates the expression of mChartreuse, a green fluorescent protein, signaling the presence of inorganic mercury.

   • Construct 3: Detoxification System
     Construct 3 contains three critical genes:
     • MerA encodes the mercury reductase enzyme that reduces Hg²⁺ to elemental mercury (Hg⁰). Anchored to the outer membrane via the DogCatcher-DogTag system, MerA detoxifies Hg²⁺ outside the cell, preventing it from entering the cytoplasm.
     • gomerB encodes alkylmercury lyase, which breaks down organic mercury (e.g., methylmercury) into Hg²⁺. This intermediate is then detoxified by MerA into Hg⁰.
     • gdh encodes glucose dehydrogenase, which provides a continuous supply of NADPH, a cofactor essential for MerA's reduction of Hg²⁺ to Hg⁰. This ensures the detoxification process continues uninterrupted.

How It Works:

1. In the Presence of Inorganic Mercury (Hg²⁺):

   The merRtn501 protein from Plasmid 2 binds to Hg²⁺, triggering the expression of mChartreuse and producing green fluorescence, signaling the presence of inorganic mercury. Simultaneously, the OmpTLOT-merA complex, formed from Plasmid 3, localizes at the bacterial membrane. The MerA enzyme, anchored via the DogCatcher-DogTag system, converts Hg²⁺ to Hg⁰, which is volatile and leaves the system without producing a signal. This ensures detoxification occurs outside the cell, protecting the bacteria from toxic mercury ions.

2. In the Presence of Organic Mercury (Methylmercury):

   The gomerB enzyme from Plasmid 3 breaks down methylmercury into Hg²⁺. This inorganic mercury is detected by the merRtn501 protein on Plasmid 2, resulting in green fluorescence. To prevent the further reduction of Hg²⁺ into Hg⁰ (which would prevent fluorescence), a TEV protease is expressed. This protease cleaves MerA, halting the conversion process and allowing the system to emit the green fluorescence signal.

This dual-plasmid system ensures the bacteria can efficiently detect and detoxify mercury. The DogCatcher-DogTag system localizes key enzymes at the membrane, allowing inorganic mercury to be detoxified without producing a signal, while organic mercury is detected via green fluorescence.

Conclusion

The iGEM Bolivia team's biosensor offers a highly selective and sensitive tool for detecting mercury in environmental samples. By employing dual strategies for detecting and eliminating inorganic mercury, the system provides excellent specificity, making it ideal for field applications where accurate mercury monitoring is essential. This design leverages innovative synthetic biology tools and modular genetic systems, positioning it as a leading solution for mercury biosensing.
This dual-plasmid system ensures the bacteria can efficiently detect and detoxify mercury. The DogCatcher-DogTag system localizes key enzymes at the membrane, allowing inorganic mercury to be detoxified without producing a signal, while organic mercury is detected via green fluorescence.

References

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