Generator

Part:BBa_K3165048

Designed by: Mohit Das   Group: iGEM19_IISc-Bangalore   (2019-10-16)
Revision as of 12:05, 20 October 2019 by Mohitdas (Talk | contribs)

NOTOC__ ccdB (L83S) under araBAD

The ccdB toxin mutated to a highly unstable state so as to be expressed by common laboratory bacterial strains under arabinose activation. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 120
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 396


Usage and Biology

Biology

Controller of Cell Division or Death B (ccdB) is the toxic component of the Eschereschia coli ccdAB anti-toxin toxin system. It is a globular, dimeric protein with 101 residues per protomer, involved in the maintenance of F plasmid in cells by a mechanism involving its binding to and the poisoning of DNA Gyrase which leads to the breaking of the double-stranded DNA in the bacteria. The L83S mutant of the wild type ccdB protein is being mutated at the 83rd amino acid residue position from Leucine to Serine in the core region of the protein.
Due to the core mutation in the wild type ccdB, the L83S mutant is highly unstable.this can be further verified by the data received from the Thermal Assay which confers it's melting point to be around ~ 420C and that it aggregates at a higher temperature.
Further, we used Top10 G (gryA) R462C which has a mutation in the Gyrase A at the 462th amino acid residue position i.e. change of arginine to cysteine which makes it resistant to recognition by ccdB (L83S).

Usage

This device can be for the production ccdB L83S in Top10 G (gryA) R462C strain of Eschereschia coli. Further, it can be used in Top10 PJAT strain with Gentamicin resistance for the production and to characterize it's effects on the bacterial population.

References

  1. Anusmita Sahoo, Shruti Khare, Sivasankar Devanarayanan, Pankaj C. Jain, and Raghavan Varadarajan Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis doi: 10.7554/eLife.09532
  2. Bharat V.Adkar, Arti Tripathi, Anusmita Sahoo, Kanika Bajaj, Devrishi Goswami, Purbani Chakrabarti, Mohit K. Swarnkar, Rajesh S.Gokhale, Raghavan Varadarajan Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing https://doi.org/10.1016/j.str.2011.11.021


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