Difference between revisions of "Part:BBa K3470005"
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<partinfo>BBa_K3470005 short</partinfo>
  
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Constitutive Promoter – RBS – MerR - PmerT promoter – RBS - MerP – RBS – MerT – RBS – MerE – RBS - MerC – RBS – GFP - Double Terminator
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==Circuit==
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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).
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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.
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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)
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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.
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References:
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'''Constitutive Promoter – RBS – MerR - PmerT promoter – RBS - MerP – RBS – MerT – RBS – MerE – RBS - MerC – RBS – GFP - Double Terminator'''
  
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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
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==Usage and Biology==
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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
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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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MerP is the periplasmic component of the Mer transport system which helps in the uptake of mercury inside the cell. It binds to a single Hg (II) ion using its two conserved cysteine residues, which define its metal-binding motif. It removes any attached ligands before passing the Hg (II) on to MerT transmembrane protein. It is the most abundantly synthesized protein in the mer operon due to its role in scavenging of Hg (II) in the periplasm (Steele, R. A., & Opella, S. J.1997). MerT is a transmembrane protein which receives mercury from MerP at its first transmembrane helix and transports it into the cytoplasm of the bacterial cell (T. Barkay et al., 2003). MerE is a transmembrane component of the mer transport system which helps in the uptake of mercury inside the cell. It helps in the transport of organo-mercury compounds. MerC is a transmembrane component of the mer transport system which helps in the uptake of mercury inside the cell. It can function without the help of MerP and has been hypothesized to be required in cases of high mercury concentration (Sone et al., 2013).
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==Proposed experimentation==
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To determine the final transport design, the team has proposed three circuits consisting of a combination of genes among MerP, MerC, MerT and MerE. The circuit showing the most effective results can be chosen as the bio-brick for the transport system for our first plasmid.
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Circuits we have proposed to test for the final transport design system:
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1. MerP - MerT - MerC - MerE,
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2. MerC - MerE
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3. MerP - MerT - MerE.
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To test the efficiency and characterize each of the 4 parts separately we carry out experiments with each of the parts making use of 2 test circuits and 2 controls.
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'''Circuits:'''
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1. The final transport design system
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2. Constitutive Promoter- RBS – (The part to be tested, i.e. MerP, MerC, MerT or MerE) -RBS-Double Terminator.  
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'''Controls:'''
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1. Final circuit design without the part to be tested
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2. Wild type ''Escherichia coli DH5alpha''. 
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''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 be centrifuged and the mercury concentration in the supernatant for each set must be determined with gas chromatography. Plots of concentration vs time for each of the sets should be analysed to understand the efficiency of the parts in transporting methylmercury.
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'''Expected result:'''
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The most efficient transport system is the final transport circuit design. Also, the team expects to see that MerC contributes significantly in the presence of other transport system elements. It shows that MerC is the most efficient tool as it is sufficient for mercurial transport into cells. The team also expects to see that MerE contributes significantly in the presence of other transport system elements but less efficient than MerT in presence of other transport system elements. What is unexpected is if there are two transport system circuits with similar efficiency, then one with the least genetic burden must be selected. The expected result must show the efficiency of MerP, MerT, MerE, and MerC all together in transporting methylmercury, which should be higher than the natural transport (without mer operon transporters).
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==Sequence and features==
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<partinfo>BBa_K3470005 SequenceAndFeatures</partinfo>
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==References==
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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
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Rossy, E., Sénèque, O., Lascoux, D., Lemaire, D., Crouzy, S., Delangle, P., & Covès, J. (2004). Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase? FEBS Letters, 575(1–3), 86–90. https://doi.org/10.1016/j.febslet.2004.08.041
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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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Steele, R. A., & Opella, S. J. (1997). Structures of the reduced and mercury- bound forms of merP, the periplasmic protein from the bacterial mercury detoxification system. Biochemistry, 36(23), 6885–6895. https://doi.org/10.1021/bi9631632

Latest revision as of 16:03, 24 October 2020

Methylmercury Transport system design-1

Circuit

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

Usage and Biology

MerP is the periplasmic component of the Mer transport system which helps in the uptake of mercury inside the cell. It binds to a single Hg (II) ion using its two conserved cysteine residues, which define its metal-binding motif. It removes any attached ligands before passing the Hg (II) on to MerT transmembrane protein. It is the most abundantly synthesized protein in the mer operon due to its role in scavenging of Hg (II) in the periplasm (Steele, R. A., & Opella, S. J.1997). MerT is a transmembrane protein which receives mercury from MerP at its first transmembrane helix and transports it into the cytoplasm of the bacterial cell (T. Barkay et al., 2003). MerE is a transmembrane component of the mer transport system which helps in the uptake of mercury inside the cell. It helps in the transport of organo-mercury compounds. MerC is a transmembrane component of the mer transport system which helps in the uptake of mercury inside the cell. It can function without the help of MerP and has been hypothesized to be required in cases of high mercury concentration (Sone et al., 2013).

Proposed experimentation

To determine the final transport design, the team has proposed three circuits consisting of a combination of genes among MerP, MerC, MerT and MerE. The circuit showing the most effective results can be chosen as the bio-brick for the transport system for our first plasmid.

Circuits we have proposed to test for the final transport design system:

1. MerP - MerT - MerC - MerE,

2. MerC - MerE

3. MerP - MerT - MerE.

To test the efficiency and characterize each of the 4 parts separately we carry out experiments with each of the parts making use of 2 test circuits and 2 controls.

Circuits:

1. The final transport design system

2. Constitutive Promoter- RBS – (The part to be tested, i.e. MerP, MerC, MerT or MerE) -RBS-Double Terminator.

Controls:

1. Final circuit design without the part to be tested

2. Wild type Escherichia coli DH5alpha.

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 be centrifuged and the mercury concentration in the supernatant for each set must be determined with gas chromatography. Plots of concentration vs time for each of the sets should be analysed to understand the efficiency of the parts in transporting methylmercury.

Expected result:

The most efficient transport system is the final transport circuit design. Also, the team expects to see that MerC contributes significantly in the presence of other transport system elements. It shows that MerC is the most efficient tool as it is sufficient for mercurial transport into cells. The team also expects to see that MerE contributes significantly in the presence of other transport system elements but less efficient than MerT in presence of other transport system elements. What is unexpected is if there are two transport system circuits with similar efficiency, then one with the least genetic burden must be selected. The expected result must show the efficiency of MerP, MerT, MerE, and MerC all together in transporting methylmercury, which should be higher than the natural transport (without mer operon transporters).

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 954
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1897
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2624
    Illegal SapI site found at 939

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

Rossy, E., Sénèque, O., Lascoux, D., Lemaire, D., Crouzy, S., Delangle, P., & Covès, J. (2004). Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase? FEBS Letters, 575(1–3), 86–90. https://doi.org/10.1016/j.febslet.2004.08.041

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

Steele, R. A., & Opella, S. J. (1997). Structures of the reduced and mercury- bound forms of merP, the periplasmic protein from the bacterial mercury detoxification system. Biochemistry, 36(23), 6885–6895. https://doi.org/10.1021/bi9631632