Part:BBa_K346003:Design
RBS(B0032)+MBP(mercury metal binding peptide engineered from MerR)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 184
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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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
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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).