Coding
WhT

Part:BBa_K3979005:Design

Designed by: Akshay J, Abhishek Raghunathan   Group: iGEM21_IISER_TVM   (2021-10-18)


T. aestivum Wild Type Chitinase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 712
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 7
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 73
    Illegal NgoMIV site found at 621
  • 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 II precursor from Triticum aestivum GenBank ID: AAD28730.1


References


  • 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.
  • 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
  • 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