Difference between revisions of "Part:BBa K346005:Design"

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Mercury (II) ions absorption device

Design Notes

this part is designed to combine three subparts----the T7promoter-rbs-Dsba-mbp-terminator, T7promoter-rbs-mbp-terminator and T7promoter-rbs-lpp-ompa-mbp-terminator.

Metal binding pepside(MBP)

MBP was designed as a single polypeptide that could fold into an antiparallel coiled coil, just like MerR, the mercury-responsive metalloregulatory protein MerR dose. As a result, the engineered MBP has a similar mercury binding capacity as MerR. We got the the gene of mbp by PCR with the plasmid from Anna.

Dsba-mbp

Dsba-mbp is a fusion protein aiming to transport the MBP protein to the periplasm. Dsba is a signal peptide, which can be recognized and transported to the periplasm.

Lpp-ompa-mbp

Lpp-ompa-mbp is designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide(MBP). The signal peptide of the N-termini of this fusion protein targets the protein on the membrane while the trans-membrane domain of Ompa serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane.

Experiment:

The three subparts are ligated together step by step with sub-clone. To test the function of the device, both expression experiment and function test is necessary. As a result, we have test the size of the expressed proteins with SDS-page and Western blot. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES, which can test the quantity of mercury binding by the bacteria with the device.

Results:

Expression of proteins

The Dsba-mbp, mbp and lpp-ompa-mbp are inserted into the commercial plasmid PET21A. Then the plasmid is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot of the expressed proteins in these three parts(figure2) show that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD for MBP, ~40kD for Dsba-MBP and ~27kD for LPP-OMPA-MBP, all of which are consist with the predicted size, indicating that all these three coding sequence can be expressed normally in the right place.

Function test

Having made sure that the protein can express normally in the proper place, the function tests experiment are carried out with ICP-AES. To test the efficiency of mercury absorption of our mercury bioabsorption device in different concentration of mercury, the concentration gradient is set from 10^-7M to 10^-5M, the results are shown in figure 3. It is obvious that the efficiency of this mercury absorption device increases with the increase of the mercury concentration and it can binding Hg(II) with high efficiency and high sensitivity from the concentration of 10^-6 M compared to that of the control. In addition, compare the capacity of metal binding of the device which contains three subparts with the subparts alone(MBP, Dsba-MBP and LPP-OMPA-MBP), these four parts are tested in the mercury concentration of 10^-5M to compare with each other, with the results shown in figure 4. It is necessary to point that that the device consisting of the three subparts seems to be less efficient than that of the surface display part: lpp-ompa-mbp though it is better than the mbp and Dsba-mbp. This “unusual” phenomenon can be explained as that with the number of exogenous protein increases, the efficiency of expression of protein decreases quickly, for the hard burden due to these proteins.

Source

MerR is from plasmid NR1 lpp-ompa-mbp is from plasmid pASK-IBA3 both these two plasmids are offered by Anne O. Summers.

References

[1]Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.

[2]Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.

[3]Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. ProcNatl Acad Sci U S A 90, 10444–10448

[4]Daugherty, P. S., Olsen, M. J., Iverson, B. L. & Georgiou, G. (1999).Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng 12, 613–621.

[5]Song, L., Caguiat, J., Li, Z., Shokes, J., Scott, R. A., Olliff, L. &Summers, A. O. (2004). Engineered single-chain, antiparallel,coiled coil mimics the MerR metal binding site. J Bacteriol 186,1861–1868.

[6]Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719