Part:BBa_K3470021

Circuit

Constitutive Promoter - RBS - MerE - RBS - Double Terminator

=Usage and Biology

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. (Sone et al., 2013)

Proposed Experimentation

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:

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 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.

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