Coding

Part:BBa_K2929004:Design

Designed by: James Hammond   Group: iGEM19_St_Andrews   (2019-10-11)


mOrange + SpyTag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 820
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 594
    Illegal AgeI site found at 706
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Besides codon-optimising the sequence for our E. coli chassis, we used a protein tertiary structural predictor to assess if the protein folds correctly. The predictor suggested that the protein folds correctly, having a similar topology to mOrange (PDB ID: 2H5O). This suggested to us that the SpyTag would have no negative impact on the fluorescence of the part.


Added flanking 5' NcoI and 3' BamHI cut sites to allow cloning into our pEHisTEV vector. Added extra Glycine residue 5' to 2xGGSG linker.

Source

Obtained the sequences for BBa_E2050 and BBa_K1159201 from the registry, then joined the sequences via a 2xGGSG linker and ordered sequence from IDT. The sequence was codon-optimised for E. coli using the IDT codon optimisation tool.

References