Coding
ChiA

Part:BBa_K3979010:Design

Designed by: Akshay J, Abhishek Raghunathan   Group: iGEM21_IISER_TVM   (2021-10-19)
Revision as of 23:47, 20 October 2021 by Asmita 05 (Talk | contribs) (→‎References)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


ChiA is from Amycolatopsis orientalis strain B-37


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 63
    Illegal NheI site found at 81
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 7
    Illegal XhoI site found at 970
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 760


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 of Amycolatopsis orientalis GenBank ID: WP_044850297.1


References

  1. Streptomyces orientalis: Yoshio Tominaga & Yoshio Tsujisaka (1976) Purifications and Some Properties of Two Chitinases from Streptomyces orientalis Which Lyse Rhizopus Cell Wall, Agricultural and Biological Chemistry, 40:12, 2325-2333, DOI: 10.1080/00021369.1976.10862407
  2. Murata T, Amarume S, Hattori T, et al. Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity. Biochemical and Biophysical Research Communications. 2005;336(2):514-520. doi:10.1016/j.bbrc.2005.08.123
  3. Saima, & Kuddus, Mohammed & Roohi, & Ahmad, Iffat. (2013). Isolation of novel chitinolytic bacteria and production optimization of extracellular chitinase. Journal of Genetic Engineering and Biotechnology. 11. 39–46. 10.1016/j.jgeb.2013.03.001
  4. 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