Coding

Part:BBa_K4212004:Design

Designed by: Shirin Bamezai   Group: iGEM22_Imperial_College_London   (2022-09-29)


CotZ_ChiS


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 705
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 705
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 705
    Illegal BglII site found at 48
    Illegal BamHI site found at 690
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 705
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 705
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Selection of CotZ enables display on spore crust rather than a midspore localisation, however abundance is lower than other anchor proteins. This influences the activity of observed of both the chitinase enzyme and the level of chitin degradation obtained through the engineered spore. Chitinase sequence is codon optimised for B. subtilis.


Source

The CotZ CDS comes from the genome of B. subtilis, and was produced through gene synthesis. The chitinase protein sequence comes from the B. pumilus, however this was codon-optimised for B. subtilis and synthesized as a gBlock (IDT). The individual CDSs were also ordered with Type IIS restriction sites on either end, to enable scarless fusion as a single CDS in a level 0 plasmid within a Golden Gate system.

References