Generator

Part:BBa_K3962356:Design

Designed by: Siheng Li   Group: iGEM21_Leiden   (2021-10-13)


Constant expression of antitoxin RelB by constitutive promoter J23117


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 XhoI site found at 164
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

BBa_J23117 has relatively low expression avoiding metabolic burden. 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 90 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 sequences of subparts are from iGEM registry.


References