Part:BBa_K3470005

Circuit

Constitutive Promoter – RBS – MerR - PmerT promoter – RBS - MerP – RBS – MerT – RBS – MerE – RBS - MerC – RBS – GFP - Double Terminator

Usage and Biology

MerA encodes the mercury reductase enzyme. It reduces Hg (II) to relatively inert and volatile Hg (0) in an NADPH dependent reaction. (Parks et al., 2009) MerB encodes the organomercurial lyase enzyme and is usually found immediately downstream to MerA. It catalyzes breaking the bond between carbon and mercury through the protonolysis of compounds such as methylmercury. This produces the less mobile Hg (II) which is then reduced to Hg (0) by MerA. (Miki et al., 2008).

Proposed experimentation

Methylmercury concentrations in the presence and absence of MerA and MerB must be checked with 3 circuits. The first with the presence of both MerA and MerB, the second and third with deletion of MerA and MerB respectively and the control with the absence of both MerA and MerB. An increase in the Mer spectrum with the introduction of MerB and MerA must be mapped where expected conclusion is that the addition of the two genes confers to better resistance to methylmercury. The team performed the MTT assay to map the resistance provided by each gene MerA and MerB.

The principle of the MTT assay is that for most viable cells mitochondrial activity is constant and thereby an increase or decrease in the number of viable cells is linearly related to mitochondrial activity. Thus, any increase or decrease in viable cell number can be detected by measuring formazan concentration reflected in optical density (OD) using a plate reader at 540 and 720 nm. For drug sensitivity measurements, the OD values of wells with cells incubated with drugs are compared to the OD of wells with cells not exposed to drugs. (Van Meerloo, Kaspers and Cloos, 2011)

Quantitative mapping of the resistance provided by each gene must be mapped using the graphs. The introduction of MerB and MerA must increase the Mer spectrum. The resistance provided is expected to be in the order Control<Circuit 3< Circuit 2< Circuit 1. Hence the addition of the two genes confers better resistance to methylmercury.

References

Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122

Parks, J. M., Guo, H., Momany, C., Liang, L., Miller, S. M., Summers, A. O., & Smith, J. C. (2009). Mechanism of Hg-C protonolysis in the organomercurial lyase MerB. Journal of the American Chemical Society, 131(37), 13278–13285. https://doi.org/10.1021/ja9016123

van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitivity assays: the MTT assay. Methods in molecular biology (Clifton, N.J.), 731, 237–245. https://doi.org/10.1007/978-1-61779-080-5_20