Part:BBa_K142002:Design

lacI IS mutant (IPTG unresponsive) T276A

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 mutants presented derive from the BioBrick holding lacI by itself (C0012). Site-directed mutagenesis was performed by PCR, subsequent DpnI digest and transformation. The following primers were used:

R197F forward

CGGCGCGTCTGTTTCTGGCTGGCTG

R197A forward

CGGCGCGTCTGGCGCTGGCTGGCTG

R197F reverse

CAGCCAGCCAGAAACAGACGCGCCG

R197A reverse

CAGCCAGCCAGCGCCAGACGCGCCG

T276F forward

GGATACGACGATTTTGAAGACAGCTC

T276A forward

GGATACGACGATGCGGAAGACAGCTC

T276F reverse

GAGCTGTCTTCAAAATCGTCGTATCC

T276A reverse

GAGCTGTCTTCCGCATCGTCGTATCC

All lacI IS holding BioBricks were verified by sequencing.

References

(1) Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247-54.

(2) Friedman, A. M., Fischmann, T. O., and Steitz, T. A. (1995) Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science 268, 1721-7.

(3) Suckow, J., Markiewicz, P., Kleina, L. G., Miller, J., Kisters-Woike, B., and Muller-Hill, B. (1996) Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J Mol Biol 261, 509-23.