Overview

Our mercury biosensor aims to provide an innovative tool for the rapid and precise detection of mercury in water, with a particular focus on organic mercury. By leveraging genetically engineered bacteria, we sought to create a biosensor capable of identifying toxic concentrations of mercury in environmental settings, specifically in aquatic ecosystems.

To achieve this, we employed the design-build-test-learn (DBTL) cycle, a fundamental framework in synthetic biology that allows for continuous optimization throughout the development process. This iterative approach was instrumental in refining each stage of the biosensor’s evolution.

Design

The iGEM Bolivia team developed two innovative strategies for creating a biosensor capable of selectively detecting both organic and inorganic mercury. These designs incorporate genes and promoters optimized for compatibility with E. coli DH5α, a derivative of E. coli K12 (Zhang, W., et al., 2023). Our choice of strain was informed by prior success with similar bacterial systems, such as E. coli BL21 (Amarelle, V., et al., 2023), ensuring both efficiency and compatibility.

Strategy 1: “NOT Logic Gate” for Selective Detection

This strategy utilizes a "NOT logic gate" mechanism to differentiate between organic and inorganic mercury, ensuring that the biosensor is selectively responsive to organic mercury, while inhibiting responses to inorganic mercury.

Dual-Plasmid System: The bacterium is transformed with constructs  #1 and #2.
In the Presence of Organic Mercury: The protein MerRm4-1, a mercury-sensitive transcription factor, binds to organic mercury, activating the expression of the mRFP_Magenta reporter, which emits magenta fluorescence as an indicator of organic mercury.

In the Presence of Inorganic Mercury (Hg²⁺): Inorganic mercury binds to the merRtn501 transcription factor, which triggers the expression of the mChartreuse reporter, emitting green fluorescence. While inorganic mercury can also bind to MerRm4-1, the reporter is degraded due to a proteolysis mechanism controlled by the TEV enzyme, this enzyme exposes a degradation tag. At the same time the expression of the magenta reporter is suppressed by tetR, a repressor protein encoded by the tetR gene. TetR binds to the tetO site, effectively preventing mRFP_Magenta expression and ensuring that no magenta signal is emitted in response to inorganic mercury.

This strategic design guarantees that the biosensor will exclusively emit a signal in the presence of organic mercury, while any response to inorganic mercury either results in green fluorescence or is completely blocked by TetR-mediated repression.

Strategy 2: Selective Mercury Detection via Volatilization of Inorganic Mercury.

This strategy volatilizes inorganic mercury out of the cell before it enters the biosensor, ensuring that the biosensor responds selectively to organic mercury.

Dual-Plasmid System: The bacterium is transformed with Constructs  #2 and #3.
In the Presence of Inorganic Mercury (Hg²⁺):Two key protein complexes, OmpTLOT-gdh and OmpTLOT-merA, form on the bacterial membrane. Together, these complexes prevent inorganic mercury from entering the cell. The OmpTLOT-merA complex reduces Hg²⁺ to Hg⁰ (elemental mercury), a volatile form that leaves the system without eliciting a detection signal.

In the Presence of Organic Mercury The gomerB enzyme cleaves methylmercury, a form of organic mercury, converting it into inorganic mercury. This is then detected by the merRtn501 transcription factor, which activates the mChartreuse reporter, emitting green fluorescence. Additionally, TEV protease is expressed to cleave MerA, preventing further reduction of inorganic mercury and ensuring proper detection..

This strategy is particularly effective in environments where both forms of mercury are present, as it allows for the selective detection of organic mercury while volatilizing inorganic mercury to eliminate interference.

Build

Developing the Mercury Biosensor

The build phase focuses on the construction and assembly of the necessary DNA constructs to create a portable, sensitive, and selective system for detecting both organic and inorganic mercury. This phase is critical for ensuring that the biosensor meets our rigorous standards for accuracy, modularity, and safety.

Key Constructs and Components

Our biosensor design revolves around three distinct plasmid constructs, each serving a specific role in mercury detection and detoxification:
Construct 1: Encodes the merRm4-1 transcription factor, which is sensitive to both methylmercury and inorganic mercury. In the presence of organic mercury, this construct triggers the expression of mRFP_Magenta, producing magenta fluorescence.

Construct #1 (1459 pb)

Construct 2: Contains merRtn501, a transcription factor that specifically responds to inorganic mercury (Hg²⁺), inducing the expression of mChartreuse, which emits green fluorescence. TetR is also expressed to suppress the magenta signal, ensuring clear differentiation between organic and inorganic mercury.

Construct #2 (2978 pb)

Construct 3: Comprises MerA, gomerB, and gdh for detoxification. MerA reduces Hg²⁺ to Hg⁰, while gomerB breaks down methylmercury into Hg²⁺. gdh provides NADPH for the reduction process.

Construct #3 (4603 pb)

These constructs were synthesized by IDT and Twist. They will be transformed into E. coli DH5α, with combinations of plasmids 1 with 2, and 3 with 2. Although this requires the use of two selection markers, it avoids the need for molecular cloning, simplifying the process.

TEST

Sensitivity Evaluation

To ensure the selective detection of organic mercury, we will define the maximum allowable concentration of methylmercury at 1.6 µg/L or 1.6 ppb, aligning with the World Health Organization (WHO) guidelines for 2021. The sensitivity of our biosensor, designed according to the promoter characteristics outlined by Zhu et al. (2023), has demonstrated a detection limit (LOD) of 0.098 µg/L for methylmercury—significantly below the WHO's recommended threshold. We will conduct tests using standard solutions of methylmercury at concentrations ranging from 0 to 100 ppb (0, 20, 40, 60, 80, 100). Before these tests, we will evaluate cell densities at OD600 as per the methodology of Guo et al. (2021). This approach is designed to capture data at concentrations close to the expected detection limits and will be supported by the development of a calibration curve using these varied standard solutions.

Evaluation of specificity to mercury and different standard metal solutions.

Detection of Methylmercury in Environmental Samples.

Upon establishing the necessary parameters for effective function, the biosensor will be employed to detect methylmercury within environmental water samples. We will utilize GFP fluorescence as an indicator within these samples, employing both positive and negative controls with predefined methylmercury concentrations to affirm the biosensor's accuracy. Exposure to real sample environments will allow us to derive mercury concentrations from established standard curves. Additionally, for a thorough comparative analysis, we will measure the total mercury concentration present in these samples using atomic absorption spectroscopy, ensuring a comprehensive understanding of mercury levels in natural water bodies.

Detection of heavy metals and measurement of gfp protein fluorescence.

Biosensor characterization

The fluorescence of each biosensor, exposed to varying concentrations of organic and inorganic mercury, will be measured to evaluate its performance.

Strategy 1: Utilizes a NOT logic gate for selective detection
Strategy 2: Employs cell surface display with mercuric reductase

New composite part characterization

The new composite part expresses both mercuric reductase (MerA) and glucose-1-dehydrogenase (GDH) on the bacterial surface. MerA requires NADPH as a cofactor, which will be provided by GDH. Characterization will occur in two phases:

• Phase 1.- Each enzyme, MerA and GDH, will be characterized individually and in combination.
• Phase 2. - The entire enzyme system will be characterized together.

Phase 1: Enzyme Characterization

Expected results. - GDH activity should increase absorbance by generating NADPH, while MerA will decrease absorbance by consuming NADPH. If both enzymes are balanced in the bacterial cultures, there should be no significant change in absorbance, indicating equilibrium between NADPH production and consumption.

Phase 2: Complete System Characterization

The full enzyme system will be assessed for its ability to reduce inorganic mercury in the presence and absence of NADPH. After culturing, aliquots will be collected and mercury concentrations in the supernatant will be quantified.

Expected results. - E. coli expressing the composite part, when cultured with glucose, should show maximum inorganic mercury reduction. Comparable results are expected with NADPH supplementation alone. No reduction should occur in the absence of glucose or NADPH, as MerA will degrade intracellularly.

NADPH recycling using surface displayed GDH and MerA on E. coli

Lessons Learned

Our most complex part (BBa_K5257023) performs the most important task for strategy 2, volatilizing inorganic mercury Hg2+ to prevent it from entering the bacteria and giving it the ability to selectively target organic mercury, without the need for NADPH cofactors.
Due to its complexity, we propose 3 redesign approaches for our new composite part (BBa_K5257023):

1. Redesign RBS30 and 31.- The stronger RBS (RBS30) controls translation of the MerA enzyme and RBS31 controls GDH. The MerA_2D variant used has a better affinity for mercury and better catalytic activity, so it consumes much more NADPH than other MerA enzymes. We believe that reversing the strengths of the RBSs could result in higher GDH production and more efficient NADPH recycling.
2. Redesign of gdh dehydrogenase. - GDH provides NADPH cofactors to the MerA enzyme, however, it makes use of glucose to function, which may hinder the use of other promoters such as PBAD by catabolic repression, it is also worth mentioning that GDH is a huge homotetramer of more than 100kDa which may hinder its anchoring. We consider that other alternatives such as glutamate dehydrogenase (Gldh, Liang et al. 2015) or alcohol dehydrogenase (ADH, Tian et al. 2022) result viable options to fulfill the same role without the use of glucose.
3. Redesign of MerA anchor. - MerA is on the bacterial surface, another alternative would be to secrete the MerA enzyme by replacing DogTag with a signal peptide, this would not limit the amount of proteins that are located within the outer membrane boundaries (Liu et al. 2016).

References