Team:MIT MAHE/Design

Project Design | iGEM MIT_MAHE

Project Design

This page describes how our project works.


To tackle our problem statement of methylmercury poisoning, we have proposed a dual plasmid system [Composite Bio-Brick 1 (Plasmid 1) and Composite Bio-Brick 2 (Plasmid 2)] with Escherichia coli Nissle 1917, a non-pathogenic, highly studied probiotic strain as the chassis.

Plasmid 1

Plasmid 1 includes a modified mer operon consisting of control, transport and enzymatic components as well as the partial control mechanism for Plasmid 2 and a reporter gene on a pSB3K5 vector. A constitutive promoter ensures continuous transcription of the homodimer MerR protein which acts like a repressor molecule in the absence of mercury. It binds to PmerT (a mercury responsive promoter) preventing transcription of the genes downstream to it. When the Hg cation is available (in the gut), it diffuses into the probiotic cell and binds to MerR which enables the transcription of all the downstream elements as the RNA polymerase can now bind to PmerT Brown et al., 2003 Nakaya et al., 1960 Park et al., 1992 Ralston & O'Halloran, 1990.

Plasmid 1 Diagram

Figure 1: Plasmid 1 Diagram

Four transport elements, MerP a periplasmic protein component along with the transmembrane proteins MerE, MerT and MerC, helps in bringing the bulk of MeHg inside the cell Barkay et al., 2003. However, the efficiency of these genes in different combinations must be checked, considering the possibility of reduced genetic burden during production (For more details - refer to experimentation section). The dual enzyme system, consisting of MerB (Organomercurial Lyase) and MerA (Mercuric (II) Reductase) are present downstream to it. MerB facilitates the breaking of the bond between organic carbon and mercury after which MerA reduces the Hg cation formed to elemental mercury Mathema et al., 2011 Parks et al., 2009.

Plasmid 2

Plasmid 2 contains the anti-inflammatory genes that must be activated only in conditions of methylmercury induced inflammations. How do we make sure this happens?

Plasmid 2 Diagram

Figure 2: Plasmid 2 Diagram

The SoxR gene which enables the transcription of SoxS promoter is present in Plasmid 1 and hence is transcribed only when Hg is present as it is present downstream to MerR. Nitric oxide, a pro-inflammatory signal that is released during inflammatory conditions binds to SoxR. This complex activates SoxS promoter on Plasmid 2 transcribing the genes downstream to it Hidalgo et al., 1998. The anti-inflammatory protein produced is Interleukin-10 (IL-10). In order to enable secretion, IL-10 has been fused with Hemolysin-A (HlyA) a transport signal peptide. Hemolysin-B (HlyB), Hemolysin-D (HlyD) and TolC enable the transport of any protein fused to HlyA (IL-10) outside the cell Gentschev et al., 2002.

Super Yellow Fluorescent Protein (SYFP) and Green Fluorescent Protein (GFP) would act as reporter genes enabling the assessment of the functioning of the Plasmid 1 and 2, respectively.


In unison, the two plasmids will break the bond of methylmercury, convert mercury cation to its elemental form and combat the side effect of mercury induced inflammation in the gut.

Video 1: Working of Plasmid 1
Video 2: Working of Plasmid 2


  1. 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.

    CrossRefGoogle ScholarBack to text
  2. Nakaya, R., Nakamura, A., & Murata, Y. (1960).

    Resistance transfer agents in Shigella.

    Biochemical and Biophysical Research Communications 3(6), 654-659.

    CrossRefGoogle ScholarBack to text
  3. Park, S. J., Wireman, J., & Summers, A. O. (1992).

    Genetic analysis of the Tn21 mer operator-promoter..

    Journal of Bacteriology 174(7), 2160-2171.

    CrossRefGoogle ScholarBack to text
  4. Barkay, T., Miller, S. M., & Summers, A. O. (2003).

    Bacterial mercury resistance from atoms to ecosystems.

    FEMS Microbiology Reviews 27(2-3), 355-384.

    CrossRefGoogle ScholarBack to text
  5. Ralston, D. M., & O-Halloran, T. V. (1990).

    Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex..

    Proceedings of the National Academy of Sciences 87(10), 3846-3850.

    CrossRefGoogle ScholarBack to text
  6. Mathema, V. B., Thakuri, B. C., & Sillanp , M. (2011).

    Bacterial mer operon-mediated detoxification of mercurial compounds: a short review.

    Archives of Microbiology 193(12), 837-844.

    CrossRefGoogle ScholarBack to text
  7. 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.

    Mechanism of Hg C Protonolysis in the Organomercurial Lyase MerB.

    Journal of the American Chemical Society 131(37), 13278-13285.

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  8. Hidalgo, E. (1998).

    The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator.

    The EMBO Journal 17(9), 2629-2636.

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  9. Gentschev, I., Dietrich, G., & Goebel, W. (2002). The E. coli -hemolysin secretion system and its use in vaccine development. Trends in Microbiology, 10(1), 39-45.

    The E. coli -hemolysin secretion system and its use in vaccine development.

    Trends in Microbiology 10(1), 39-45.

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Manipal Institute of Technology, Manipal

Manipal Academy of Higher Education

Eashwar Nagar, Manipal, Udupi, Karnataka, India