The iGEM Bolivia team proposes a workflow to characterize and validate the organic mercury biosensor

General scheme of the steps to characterize and validate our biosensor.

Characterization of each construct

Expected results. - Expected results: Each construct has a HisTag-tagged protein with a size of 30 kDa, will be purified with a kit (His-Spin Protein Miniprep, #P2001, zymoresearch) and SDS page electrophoresis will be performed, bands are expected to be present in the presence of mercury (construct #1 & #2) and IPTG (construct #3). Only construct #3 should not be induced by mercury because it does not present mercury sensors, while construct #1 and #2 will exhibit fluorescence in the presence of mercury.

Expected results of purification and electrophoresis of the proteins of each construct.

Characterization of each strategy

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

Curves of expected responses for each strategy.

Characterization of the new composite part of strategy 1

Expected results. - The new composite part BBa_K5257025 makes use of the magenta reporter coupled to a NOT logic gate circuit, allowing control of transcription and the presence of the reporter. Other iGEM teams have already characterized regulation by TetR and TEV, so we set out to demonstrate that the TEV protease is capable of cutting the magenta reporter and allowing its rapid degradation.

New composite part for strategy 1, BBa_K5257025

Purification and electrophoresis of the reporter will be carried out, it is expected that in the presence of TEV protease 2 bands will be generated, while in its absence there will be only 1 band. At the same time, construct #1 will be transformed into an E. coli DH5α with a plasmid expressing the TEV protease, it is expected that at higher concentrations of TEV there will be a lower fluorescence.

Expected results of the NOT logic gate for magenta reporter

Characterization of the new composite part of strategy 2

The proposed new composite part (BBa_K5257023), visualizes MerA and GDH enzymes on the bacterial surface, therefore we employ the LOT anchor motif which is suitable for large molecular size proteins (Liang et al. 2021), LOT can posttranslationally couple with both enzymes thanks to the Dogtag/DogCatcher system within minutes (Keeble et al. 2022). To avoid the need to add NADPH cofactors we expect to generate an NADPH recycling system via GDH (Tian et al. 2022). Therefore, we will first measure the enzymatic activity of MerA and GDH, then evaluate the ability to recycle NADPH.

Theoretical basis of the new composite part for strategy 2

Expected results. - The enzymatic activity of GDH is expected to cause an increase in absorbance, while MerA will consume NADPH, resulting in a decrease in absorbance. If the two bacterial strains are grown in the correct proportions, no significant change in absorbance should occur, indicating a balance between NADPH production and consumption.

Expected results. - E. coli cells expressing the new composite part, when cultured with glucose, should demonstrate the maximum rate of inorganic mercury reduction. Similarly, the presence of NADPH alone should yield comparable results. In the absence of glucose or NADPH, no reduction of inorganic mercury is expected, as MerA will degrade intracellularly.

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

Expected results of NADPH recycling of the new composite part of strategy 2

Biosensor validation

Traditionally MerA and MerB enzymes were used as a bioremediation system, our strategy 2 proposes to use the MerA enzyme on the surface to volatilize Hg2+ and reduce its bioavailability, favoring only organic mercury to generate a measurable fluorescent response. In contrast, strategy 1 mutes the signal emitted by organic mercury when inorganic mercury is present. Therefore, we will subject each biosensor to different similar heavy metals such as cadmium or lead to see their selectivity to organic mercury, we do not expect fluorescence against other metals, because merR from Tn501 and the m4-1 mutant exhibit high selectivity (Wang et al. 2020).

Validate with environmental samples

We will validate our biosensors by determining the amount of organic mercury in real samples with concentrations already known by atomic absorption spectroscopy (AAS), we expect to obtain a high correlation between both methods. We expect a high sensitivity of both strategies (ppb), because organic mercury is more liposoluble than inorganic mercury.

Expected correlation between both strategies of our biosensor with atomic absorption spectroscopy (AAS).

On-Hardware Validation

We propose to deploy our biosensors in a portable, cheap and easy to use platform. To keep the bacteria alive and active we will use lyophilization or alginate encapsulation (see lab-experiments ), our biosensor will be inside autoclavable glass tubes with a low volume requirement (1-2ml) of sample.

While incubation is required, the mChartreuse reporter is fast maturing and will provide results in less time than the mRFPmagenta reporter (t= 18 to 24h, Bao et al. 2020 & Fraikin et al. 2024). Therefore, we expect that strategy 2 of our biosensor will give results in less time than strategy 1, thus reducing the requirements before measuring fluorescence in the hardware.

A calibration curve will be performed with known mercury concentrations before determining the concentration of mercury present in the samples. The excitation wavelength of the mChartreuse is λex=487nm and the emission wavelength is λem=510nm, while mRFPmagenta has λex=588nm and λem=616nm. After data collection, the bacteria will be inactivated by taking them to the autoclave and will be followed as presented in “Waste and Disinfection Protocol for the Mercury Biosensor Device” (Safety ).

Sequence of steps to validate our biosensor in hardware.

References