Composite

Part:BBa_K3470004

Designed by: Shravan Balasubramaniam   Group: iGEM20_MIT_MAHE   (2020-10-17)

Modified mer Operon

Circuit

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 a GFP reporter downstream to all coding regions and control elements.

Usage and Biology

We have proposed a constitutive promoter to ensure 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 et al., 2013). MerA and MerB is proposed as 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 have also proposed a mechanism to tackle that problem: A Composite BioBrick (2) having the anti-inflammatory cytokine, IL-10 and the associated transport and regulatory system. GFP must be used to assess the functioning of circuit components, mainly the MerR regulation. (Kremers et al., 2006)

Proposed experimentation

To check the threshold of mercury required by the transformed bacteria to generate a response, this circuit vs the control- Constitutive Promoter – GFP - Double Terminator should be compared. E. coli cells inoculated with methylmercury chloride must be 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 must then be 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 must be analysed to understand the efficiency of the parts in transporting methylmercury. A plot of OD as a function of time at different methylmercury concentrations and a plot of GFP fluorescence intensity as the function of time, keeping methylmercury concentration constant must be mapped. The threshold of response must be 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 652
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1878
    Illegal NgoMIV site found at 3162
    Illegal NgoMIV site found at 3210
    Illegal NgoMIV site found at 3272
    Illegal NgoMIV site found at 3483
    Illegal NgoMIV site found at 4088
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4982
    Illegal SapI site found at 637

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

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