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Part:BBa_K3962348:Design

Designed by: Siheng Li   Group: iGEM21_Leiden   (2021-10-12)
Revision as of 08:03, 16 October 2021 by SihengLi (Talk | contribs)

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J23113 promoter regulated constant expression of toxin RelE


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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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. For Hok, it has also been used as a part of a kill switch. 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 RelE in it. The total length of the forward primer was 83 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