Coding

Part:BBa_K533006:Design

Designed by: ZHANG Yunxiao   Group: iGEM11_Tsinghua   (2011-09-29)
Revision as of 16:08, 5 October 2011 by Wishyx (Talk | contribs) (→‎References)

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Multi-Proline-mCherry


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 109
    Illegal BamHI site found at 908
    Illegal XhoI site found at 113
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The coding sequence is driven by T7 promoter.

We added his tag to the N-terminal to facilitate purification of the expressed protein. Proline rich sequence put here is much longer than the length required for SH3 binding, for tighter binding to SH3 domain.

Proline rich sequence PVPPPVPPRRRP is from the structure of SH3 complexed with a proline rich peptide solved by Wittekind, et al. The Kd value is determined by them to be 3.57uM.

Source

mCherry sequence is from Clontech plasmid pmCherry-N1 and multi-proline sequence is synthesized from a consensus sequence of SH3 binding sequence.

References

Solution structure of the grb2 n-terminal sh3 domain complexed with a ten-residue peptide derived from sos: direct refinement against noes, j-couplings and 1h and 13c chemical shifts. Wittekind M, Mapelli C, Lee V, Goldfarb V, Friedrichs MS, Meyers CA, Mueller L J.Mol.Biol. (1997) 267 p.933