Part:BBa_K1980001:Design

TAT Copper Storage Protein 1 sfGFP

Assembly Compatibility:

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

  Illegal XhoI site found at 886
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 448
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The part is codon optimised for E. coli. A native E. coli TAT sequence has been added 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 flexible linker (GlySerGlySerGlySer) added between Csp1 and the sfGFP in order to allow the Csp1 and sfGFP to fold separately. There is a hexahistidine purification tag on C-terminus in order to purify the protein whilst preserving the N-terminal TAT sequence.

Source

The source organism for the main protein sequence is Methylosinus trichosporium OB3b. The TAT sequence originates from E. coli multi copper oxidase enzyme CueO. We ordered it as codon optimised DNA from IDT.

References

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

TAT sequences from: http://www.lifesci.dundee.ac.uk/groups/tracy_palmer/docs/signals.htm