Other

Part:BBa_K1980010:Design

Designed by: Sam Garforth   Group: iGEM16_Oxford   (2016-10-11)


pCopA TAT Csp1 sfGFP with divergent CueR


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 438
    Illegal NheI site found at 461
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1466
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1028
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The part is codon optimised for E. coli. A native E. coli TAT sequence has been added to the Csp1 in place of the original host TAT sequence in an attempt to make the protein go to the periplasm. The E. coli TAT sequence from CueO in particular was chosen only because it is quite short and CueO is also involved in copper homeostasis. There is a hexahistidine purification tag on C-terminus of Csp1 in order to purify the protein whilst preserving the N-terminal TAT sequence.

The linker between Csp1 and sfGFP is short and hydrophilic (GlySerGlySerGlySer) to allow the protein domains to fold separately.

CueR is expressed divergent form the pCopA promoter.

Source

The source organism for the main protein sequence of TAT Csp1 is Methylosinus trichosporium OB3b. The TAT sequence originate from E. coli multi-copper oxidase enzyme CueO. The CueR and pCopA components of the promoter also originate from E. coli. We ordered this part as codon optimised DNA from IDT.

References

Danya J. Martell, Chandra P. Joshi, Ahmed Gaballa, Ace George Santiago, Tai-Yen Chen, Won Jung, John D. Helmann, and Peng Chen (2015) “Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription” Proc Natl Acad Sci U S A. 2015 Nov 3; 112(44): 13467–13472.

Yamamoto K, Ishihama A. (2005) “Transcriptional response of Escherichia coli to external copper.” Mol Microbiol. 2005 Apr;56(1):215-27.

Nicolas Vita, Semeli Platsaki, Arnaud Basle, Stephen J. Allen, Neil G. Paterson, Andrew T. Crombie, J. Colin Murrell, Kevin J.Waldron & Christopher Dennison (2015) “A four-helix bundle stores copper for methane oxidation”, Nature 525 issue 7567 pg. 140-143 doi:10.1038/nature14854