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Part:BBa_K3470005

Designed by: Shravan Balasubramaniam   Group: iGEM20_MIT_MAHE   (2020-10-17)
Revision as of 09:58, 17 October 2020 by Shrav 27 (Talk | contribs)

Constitutive Promoter – RBS – MerR - PmerT promoter – RBS - MerP – RBS – MerT – RBS – MerE – RBS - MerC – RBS – GFP - Double Terminator 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). The team checked for methylmercury concentrations in the presence and absence of MerA and MerB with 3 circuits. The first with presence of both MerA and MerB, the second and third with deletion of MerA and MerB respectively and the control with absence of both MerA and MerB. The team tested to see the increase in the Mer spectrum with the introduction of MerB and MerA to conclude that the addition of the two genes confer to a 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) The team could quantitatively map the resistance provided by each gene using the graphs. The introduction of MerB and MerA increases the Mer spectrum. The resistance provided should be in the order Control<Circuit 3< Circuit 2< Circuit 1. Hence the addition of the two genes confers better resistance to methylmercury.

References:

Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews, 27(2–3), 355–384. https://doi.org/10.1016/S0168-6445(03)00046-9 

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

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