Generator

Part:BBa_K3962347:Design

Designed by: Siheng Li   Group: iGEM21_Leiden   (2021-10-12)


J23113 promoter regulated constant expression of toxin CcdB


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 727
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 177


Design Notes

J23113 promoter has low transcriptional activity. Using it to regulate the toxin gene can not only prevent high lethality, but also maintain its function. We used a PCR-based cloning method to clone the construct. This was done by fusing the sequence of the promoter to the forward primer which contains sequence annealing with biobrick prefix and an overlap sequence of the coding sequence of CcdB in it. The total length of the forward primer was 93 bp. For the reverse primer, a 22 bp primer was used that is homologous to the suffix of the biobrick. For the PCR a touch-down program was used to get a higher degree of annealing specificity due to the long length of the primers. Codon optimization was performed for the expression of the gene in E. coli.



Source

All parts come from the iGEM registry.


References