Part:BBa_K3470011

Constitutive Promoter - RBS – MerT - RBS – Double Terminator

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. 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. The team could 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, 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:

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