Difference between revisions of "Part:BBa K142000:Design"

 
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<partinfo>BBa_K142000 short</partinfo>
 
<partinfo>BBa_K142000 short</partinfo>
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===Design Notes===
 
===Design Notes===
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During ste-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.
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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.
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===Source===
 
===Source===
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R197F forward  
 
R197F forward  
 +
 
CGGCGCGTCTGTTTCTGGCTGGCTG  
 
CGGCGCGTCTGTTTCTGGCTGGCTG  
 +
 
R197A forward  
 
R197A forward  
 +
 
CGGCGCGTCTGGCGCTGGCTGGCTG  
 
CGGCGCGTCTGGCGCTGGCTGGCTG  
 +
 
R197F reverse  
 
R197F reverse  
 +
 
CAGCCAGCCAGAAACAGACGCGCCG  
 
CAGCCAGCCAGAAACAGACGCGCCG  
 +
 
R197A reverse  
 
R197A reverse  
 +
 
CAGCCAGCCAGCGCCAGACGCGCCG  
 
CAGCCAGCCAGCGCCAGACGCGCCG  
 +
  
  
 
T276F forward  
 
T276F forward  
 +
 
GGATACGACGATTTTGAAGACAGCTC  
 
GGATACGACGATTTTGAAGACAGCTC  
 +
 
T276A forward  
 
T276A forward  
 +
 
GGATACGACGATGCGGAAGACAGCTC  
 
GGATACGACGATGCGGAAGACAGCTC  
 +
 
T276F reverse  
 
T276F reverse  
 +
 
GAGCTGTCTTCAAAATCGTCGTATCC  
 
GAGCTGTCTTCAAAATCGTCGTATCC  
 +
 
T276A reverse  
 
T276A reverse  
 +
 
GAGCTGTCTTCCGCATCGTCGTATCC  
 
GAGCTGTCTTCCGCATCGTCGTATCC  
  
 
All lacI IS holding BioBricks were verified by sequencing.
 
All lacI IS holding BioBricks were verified by sequencing.
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===References===
 
===References===

Latest revision as of 14:46, 17 October 2008

lacI IS mutant (IPTG unresponsive) R197A


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

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