Coding
BaT

Part:BBa_K3979006:Design

Designed by: Akshay J, Abhishek Raghunathan   Group: iGEM21_IISER_TVM   (2021-10-18)
Revision as of 01:44, 20 October 2021 by Asmita 05 (Talk | contribs) (→‎References)


H. vulgare Wild-Type Chitinase


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 976
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1
    Illegal NgoMIV site found at 88
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

While designing the sequence of the recombinant chitinase, there were several considerations that we needed to keep in mind. Firstly, since we planned to express the chitinase in E. coli, it became necessary that the final molecular weight was less than 100 kDa to enable robust protein folding post expression in the chassis. This imposed a constraint on the size and number of wild-type domains we could join. Secondly, since we aimed to use the chitinase as a therapeutic, the activity of our enzyme as a function of temperature and pH were essential parameters to keep in mind. Our interactions with distinguished crystallographers made us realize that these parameters would depend heavily on the temperature and pH activity curves of the wild-type chitinases we chose. Hence, we decided on wild-type enzymes that retained a significant fraction of their activities at physiological temperatures and pH.



Source

Chitinase from Hordeum vulgare GenBank ID: AAA18586.1


References

  1. 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
  2. Kirubakaran SI, Sakthivel N. Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli. Protein Expr Purif. 2007 Mar;52(1):159-66. doi: 10.1016/j.pep.2006.08.012. Epub 2006 Sep 6. PMID: 17029984.