Coding

Part:BBa_K2238003:Design

Designed by: Bryan Wilkins   Group: iGEM17_ManhattanCol_Bronx   (2017-10-21)


Glucose oxidase (A. niger) Quadruple Mutant, Cysteine tagged


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 36
    Illegal BamHI site found at 346
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 370
    Illegal BsaI.rc site found at 1588


Design Notes

The amino acid sequence was converted to the E. coli optimized DNA coding sequence (IDT https://www.idtdna.com/CodonOpt). We added the coding sequence for an N-terminal His-tag and TEV protease for convenient protein isolation. This leaves a small glycine scar on the N-terminal end of the protein following enzymatic cleavage. There is a short 4-glycine-Serine tag followed by a SacI site prior to the stop codon. This was added for flexibility with potentially adding peptide and protein fusions.

The following mutations were made with consideration for codon optimization in E. coli. T56V, T132S, H469C, C543V. This parts coding is based on part BBa_K2238001 with identical sequencing prior to the CYS-tag addition.


Source

Aspergillus niger Gene Bank: AAA32695.1 (E.C 1.1.3.4) Wild type sequence. Mutations include: T56V, T132S, H469C, C543V


References

J. Todd Holland, Jason C. Harper, Patricia L. Dolan, Monica M. Manginell, Dulce C. Arango, Julia A. Rawlings, Christopher A. Apblett, Susan M. Brozik. Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability. PLoS ONE, 2012 vol. 7 (6) pp. e37924-10


J. Todd Holland, Carolin Lau, Susan Brozik, Plamen Atanassov, and Scott Banta. Engineering of Glucose Oxidase for Direct Electron Transfer via Site-Specific Gold Nanoparticle Conjugation. J. Am. Chem. Soc., 2011 vol. 133(48) pp. 19262-19265