Generator

Part:BBa_K142012:Design

Designed by: Julius Rabl   Group: iGEM08_ETH_Zurich   (2008-10-28)


lacI IS mutant R197A T276A with RBS and terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

During site-directed mutagenesis, the codons to be mutated were replaced with the most highly utilized codons in E. coli to prevent complications from the use of rare codons. The lacI IS sequences were analyzed for BioBrick restriction sites within the coding sequence to ensure their compatibility.


Source

The lacI IS mutant presented derive from the BioBrick I763026, which as it turned out during sequencing by the Caltech team, did not have a promotor. Site-directed mutagenesis was performed by PCR, subsequent DpnI digest and transformation. The following primers were used for mutagenesis:

R197F forward

CGGCGCGTCTGTTTCTGGCTGGCTG

R197A forward

CGGCGCGTCTGGCGCTGGCTGGCTG

R197F reverse

CAGCCAGCCAGAAACAGACGCGCCG

R197A reverse

CAGCCAGCCAGCGCCAGACGCGCCG


T276F forward

GGATACGACGATTTTGAAGACAGCTC

T276A forward

GGATACGACGATGCGGAAGACAGCTC

T276F reverse

GAGCTGTCTTCAAAATCGTCGTATCC

T276A reverse

GAGCTGTCTTCCGCATCGTCGTATCC


References