Coding
PC1

Part:BBa_K3979007:Design

Designed by: Akshay J, Abhishek Raghunathan   Group: iGEM21_IISER_TVM   (2021-10-18)
Revision as of 23:53, 20 October 2021 by Asmita 05 (Talk | contribs) (→‎References)

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Plant Chitinase Combo 1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal BamHI site found at 7
    Illegal XhoI site found at 1879
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1
    Illegal AgeI site found at 376
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1749


Design Notes

This is a chitinase gene designed using the catalytic domain(CD) of Triticum aestivum cultivar Sumai 3 and the chitin-binding domain (CBD) of Hordeum vulgare cultivar NK1558. we joined the sequences of both barley and wheat chitinases using common (GGGS)3 linkers. Wheat chitinase doesn’t have a chitin-binding domain, so we added barley CBD in its C-terminal in the combination. The protein sequences were linked initially and then reverse translated to nucleotide sequences(stop codon inclusive) along with codon optimization for E.coli expression using the BOOST Juggler tool. BamHI and HindIII sites were added at the 5’ and 3’ end of the gene, respectively.


Source

Chitinase II precursor from Triticum aestivum GenBank ID: AAD28730.1

Chitinase from Hordeum vulgare GenBank ID: AAA18586.1


References


  • Bartholomew ES, Black K, Feng Z, et al. Comprehensive Analysis of the Chitinase Gene Family in Cucumber (Cucumis sativus L.): From Gene Identification and Evolution to Expression in Response to Fusarium oxysporum. Int J Mol Sci. 2019;20(21):5309. Published 2019 Oct 25. doi:10.3390/ijms20215309
  • Singh A, Kirubakaran SI, Sakthivel N. Heterologous expression of new antifungal chitinase from wheat. Protein Expr Purif. 2007 Nov;56(1):100-9. doi: 10.1016/j.pep.2007.06.013. Epub 2007 Jul 12. PMID: 17697785.
  • O. Trott, A. J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010) 455-461 DOI 10.1002/jcc.21334