Coding

Part:BBa_K2934000:Design

Designed by: Nir Litver, Lior Haim   Group: iGEM19_Technion-Israel   (2019-10-07)


Glucose Oxidase-Histag A. niger optimized for B. subtilis


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 site found at 30
    Illegal BsaI site found at 361
    Illegal BsaI.rc site found at 307
    Illegal BsaI.rc site found at 1132


Design Notes

The sequence is based on the A. niger glucose oxidase (GOx) amino acid sequence and codon optimization for B. subtilis, using codon usage tables [2] [3]. The linker is based on the pBE-S commercial plasmid by TaKaRa [4] that we used for protein secretion from B. subtilis. We changed two bases at the locations 1818 and 1833 (without changing the amino acids) to removed unwanted PstI and XbaI restriction sites.

Primers for isolation of the gene (with RFC[10] suffix and prefix):

fwd: 5'-CCGCTTCTAGATGCAGACTCTCCTTGTTAGCTCAC-3'

rev: 5'-CTAGTATTAGTGGTGATGATGGTGATGTCTTGACTGG-3'

Source

The amino acid sequence was taken from UniProt [1], the gene sequence is based on the amino acid sequence. The linker and histag sequence is based on the pBE-S commercial plasmid made by TaKaRa [4].

References

[1] https://www.uniprot.org/uniprot/P13006

[2] https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=1423

[3] http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=224308&aa=1&style=N

[4] https://www.takarabio.com/products/protein-research/expression-vectors-and-systems/b-subtilis-expression-system