Device

Part:BBa_K346005:Design

Designed by: Junyi Jiao, Liang Donghai, Hu Yang&Teng Xin   Group: iGEM10_Peking   (2010-10-14)
Revision as of 23:31, 24 October 2010 by JjunyiJiao (Talk | contribs) (→‎Results:)

Mercury (II) ions absorption device


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 529
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 529
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 317
    Illegal BamHI site found at 1148
    Illegal BamHI site found at 2111
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 529
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 529
    Illegal AgeI site found at 149
  • 1000
    COMPATIBLE WITH RFC[1000]


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 construct the the gene of mbp by PCR from MerR,just as the main page shows.

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.



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