Part:BBa_K3470004

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

This circuit will be responsible for the transport of methylmercury inside the bacterial system, production of mercury (II) reductase enzyme and alkylmercurial lyase, along with its regulation. It features a modified Mer operon with an GFP reporter downstream to all coding regions and control elements. A constitutive promoter ensures the continuous transcription of MerR. When the constitutive promoter is translated, MerR gene produces a weak repressor molecule that can bind to the PmerT region, preventing the transcription of genes downstream to it (Brown et al., 2003). In the presence of Hg (II) cation, the repressor molecule would not bind to PmerT but instead, bind to mercury (II) cation and reactivate the transcription of all downstream elements - MerP, MerT, MerE and MerC to deal with the production of transport proteins that will help transport of methylmercury inside the bacterial system.(Sone, Nakamura, Pan-Hou, Sato et al., 2013). MerA and MerB produce our dual enzymes – mercuric (II) reductase and alkylmercurial lyase required for the conversion of MeHg to elemental Hg. (Mathema et al., 2011) However, since the release of elemental mercury has the potential to disturb the gut microbiota and induce inflammation, we also explore a mechanism to tackle that problem. Composite BioBrick 2 will have the anti-inflammatory cytokine, IL-10 and the associated transport and regulatory system.GFP is to assess the functioning of circuit components, mainly the MerR regulation. (Kremers et al., 2006) To check the threshold of mercury required by the transformed bacteria to generate a response, we will be using this circuit Vs the control- Constitutive Promoter – GFP - Double Terminator. E. coli cells inoculated with methylmercury chloride are grown for the required amount of time according to the results of the preliminary experiment respectively for the 2 circuits to be tested and 2 controls. The cell suspension is centrifuged and the mercury concentration in the supernatant for each set is determined with gas chromatography. Plots of concentration vs time for each of the sets are analysed to understand the efficiency of the parts in transporting methylmercury. A plot of OD in function of time at different methylmercury concentrations. A plot of GFP fluorescence intensity in function of time keeping methylmercury concentration constant. The threshold of response is noted and used as the minimum limit for the characterization experiments. Moreover, the concentrations used in the experiment are chosen through extensive literature survey and have physiological importance. However, the lower dosage limit of methylmercury cannot be precisely determined due to differences of absorption effect in different individuals.

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 Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2–3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2 Kremers, G. J., Goedhart, J., Van Munster, E. B., & Gadella, T. W. J. (2006). Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET förster radius. Biochemistry, 45(21), 6570– 6580. https://doi.org/10.1021/bi0516273 Mathema, V. B., Thakuri, B. C., & Sillanpää, M. (2011). Bacterial mer operon- mediated detoxification of mercurial compounds: A short review. Archives of Microbiology, 193(12), 837–844. https://doi.org/10.1007/s00203-011-0751-4 Sone, Y., Nakamura, R., Pan-Hou, H., Itoh, T., & Kiyono, M. (2013). Role of MerC, MerE, MerF, MerT, and/or MerP in resistance to mercurials and the transport of mercurials in escherichia coli. Biological and Pharmaceutical Bulletin, 36(11), 1835–1841. https://doi.org/10.1248/bpb.b13-00554