Device

Part:BBa_K142025:Design

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


IPTG-on tetracycline-off pulse generator with LacI mutant (R197F) and TetR expression cassette


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1360
    Illegal NheI site found at 1383
  • 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 pulse generator precursor has been assembled from tetracycline-controlled lacI generator K142046 and the constitutive TetR expression cassette I739001 using standard cloning techniques. The lacI IS mutant presented derives from site-directed mutagenesis, which was performed on the precursor K142047 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