Part:BBa_K346003:Design

RBS(B0032)+MBP(mercury metal binding peptide engineered from MerR)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 184
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in the Fig. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step.Then the constructed mbp coding sequence is inserted to the plasmid

Source

The MerR is on the plasmid NR1, which is provided by Anne O. Summers.

References

 Ralston, D. M. & Halloran, T. O. Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. Proc. Natl. Acad. Sci. 87, 3846-3850 (1990).

 Brocklehurst, K. R., Hobman, J. R., Lawley, B., Blank, L., Marshall, L. J., Brown, N. L. & Morby, A. P. ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli. Mol. Microbiol. 31, 893-902 (1999).

 Zeng, Q., Stalhandske, C., Anderson, M. C., Scott, R. A. & Summers, A. O. The core metal-recognition domain of MerR. Biochemistry 37, 15885-15895 (1998).

 Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C. E., Halloran, T. V. & Mondrago, A. Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR. Science 301, 1383-1387 (2003).

 Chen, P. R. & He, C. Selective recognition of metal ions by metalloregulatory proteins. Curr. Opin. Chem. Biol. 12,214-221 (2008).

 Shewchuk, L. M., Verdine, G. L., Nash, H. & Walsh, C.T. Mutagenesis of the cysteines in the metalloregulatory protein MerR indicates that a metal-bridged dimer activates transcription. Biochemistry 28, 6140-6145 (1989).

 Wright, J. G., Tsang, H. T., Penner-Hahn, J. E. & O’Halloran T.V. Coordination chemistry of the Hg-MerR metalloregulatory protein: evidence for a novel tridentate Hg-cysteine receptor sites. J. Am. Chem. Soc. 112, 2434-2435 (1990).