Difference between revisions of "Part:BBa K3470012"
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| + | Constitutive Promoter - RBS – MerC - RBS – Double Terminator | ||
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| + | 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) | ||
| + | To determine the final transport design, we test 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 test for the final transport design system: MerP-MerT-MerC-MerE, MerC-MerE, 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: The final transport design system, Constitutive Promoter- RBS – (The part to be tested, i.e. MerP, MerC, MerT or MerE) -RBS-Double Terminator. Controls: Final circuit design without the part to be tested, Wild type Escherichia coli DH5alpha. | ||
| + | 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. | ||
| + | Expected result: The most efficient transport system is the final transport circuit design. Also, the team could 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. What is unexpected is if there are two transport system circuits with similar efficiency, the one with the least genetic burden will be selected. The expected result should show the efficiency of MerP, MerT, MerE, MerC all together in transporting methylmercury, which should be higher than the natural transport (without mer operon transporters). | ||
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| + | References: | ||
| + | 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 | ||
Revision as of 10:48, 17 October 2020
Constitutive Promoter - RBS – MerC - RBS – Double Terminator
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)
To determine the final transport design, we test 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 test for the final transport design system: MerP-MerT-MerC-MerE, MerC-MerE, 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: The final transport design system, Constitutive Promoter- RBS – (The part to be tested, i.e. MerP, MerC, MerT or MerE) -RBS-Double Terminator. Controls: Final circuit design without the part to be tested, Wild type Escherichia coli DH5alpha. 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. Expected result: The most efficient transport system is the final transport circuit design. Also, the team could 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. What is unexpected is if there are two transport system circuits with similar efficiency, the one with the least genetic burden will be selected. The expected result should show the efficiency of MerP, MerT, MerE, MerC all together in transporting methylmercury, which should be higher than the natural transport (without mer operon transporters).
References:
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