Coding

Part:BBa_K4359001:Design

Designed by: Ira Zibbu   Group: iGEM22_IISER_TVM   (2022-08-31)


E.coli K-12 Cytolysin A (ClyA) (Y181F)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 925
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 925
    Illegal NheI site found at 4
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 925
    Illegal BglII site found at 81
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 925
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 925
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

ClyA (Y181F) is improved over its predecessor ClyA (BBa_K4359000). The N-terminal 6X His tag and thrombin recognition and cleavage site were removed.


Source

ClyA (Y181F) is a derivative of ClyA (BBa_K4359000), which is originally amplified from the genome of E.coli K12 w3110. The substitution was designed and obtained from gene synthesis.

References

[1] Kulshrestha, A., Maurya, S., Gupta, T., Roy, R., Punnathanam, S., & Ayappa, K. G. (2021). Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A [Preprint]. Biophysics. https://doi.org/10.1101/2021.10.23.465544

[2] Mueller, M., Grauschopf, U., Maier, T., Glockshuber, R., & Ban, N. (2009). The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism. Nature, 459(7247), 726–730. https://doi.org/10.1038/nature08026