Coding

Part:BBa_K4035008:Design

Designed by: Anissa Hammi   Group: iGEM21_EPFL   (2021-09-10)


Dimerization of the copper metallothionein 1 : CUP1-GGGGS(EAAAK)2GGGGS-CUP1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 391


Design Notes

The start and stop codon of the CUP1 genomic sequences were removed in order to fuse the proteins together. In addition, the full sequence was codon optimized before being ordered to avoid loops formation during synthesis. The synthetized CUP1-linker-CUP1 sequence was then inserted by Gibson Assembly in the pCTcon2-V5 plasmid (1) containing Aga2, V5 tag and the Gal1 promoter.


Source

The CUP1 sequence is the genomic sequence of the yeast copper metallothionein 1 protein (2). The sequence of the linker comes from a reverse translation of the amino acid sequence GGGGS(EAAAK)2GGGGS and was codon optimized for the yeast S. cerevisiae. The fusion protein was fully synthetized.

References

(1) Chao, G.; Lau, W. L.; Hackel, B. J.; Sazinsky, S. L.; Lippow, S. M.; Wittrup, K. D. Isolating and Engineering Human Antibodies Using Yeast Surface Display. Nat Protoc 2006, 1 (2), 755–768. https://doi.org/10.1038/nprot.2006.94.

(2) https://www.uniprot.org/uniprot/P0CX80