Coding

Part:BBa_K2934001:Design

Designed by: Nir Litver, Lior Haim   Group: iGEM19_Technion-Israel   (2019-10-08)


Invertase-Histag A. niger optimized for B. subtilis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 863
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1168
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 469
    Illegal BsaI.rc site found at 811
    Illegal SapI.rc site found at 1071


Design Notes

The sequence is based on the A. niger Invertase 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'-GATGAAGCTTCAAACGGCTTCAGTACTGCTC-3'

rev: 5'-CTAGTATTAGTGGTGATGATGGTGATGTCTTGACTGG-3'

Source

The sequence is based on the Invertase amino acid sequence [1] and is optimized for B. subtilis. The linker and histag sequence is based on the pBE-S commercial plasmid made by TaKaRa [4].

References

[1] L. M. Boddy, T. Berges, C. Barreau, M. H. Vainstein, M. J. Dobson, D. J. Ballance, j. E Peberdy. 1993. Purification and characterisation of an Aspergillus niger invertase and its DNA sequence, Current Genetics.

[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