Difference between revisions of "Part:BBa K3093000:Design"

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===Design Notes===
 
===Design Notes===
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Implying a mutation altering the substrate specificity may change the operon in the Cel+ phenotype which supports growth on cellobiose.
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ChbR is encoded within the chb operon itself. In the presence of chitobiose, ChbR binds to two direct repeats located between two NagC binding sites and activates the transcription of the chb operon by overlapping the -35 box of chbBp. In the absence of chitobiose, ChbR acts as a repressor of the chb operon. Monoacetylchitobiose-6-P has been experimentally determined to be the inducer for ChbR.
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The sequence of chbR was obtained from E.coli K12 MG1655 via PCR.Then we performed inverse PCR to achieve the site-directed mutation of amino acids of chbR. According to the literature, we chose three site-directed mutation: chbRN238S
  
 
===Source===
 
===Source===
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ChbR was obtained from the genomic sequence of Escherichia coli str. K-12 substr.MG1655
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Escherichia coli str. K-12 substr. MG1655  
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===References===
 
===References===
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[1]Plumbridge J, Pellegrini O. Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP. Mol Microbiol. 2004 Apr;52(2):437-49. PubMed PMID: 15066032.
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[2]Kachroo AH, Kancherla AK, Singh NS, Varshney U, Mahadevan S. Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose. Mol Microbiol. 2007 Dec;66(6):1382-95. Epub 2007 Nov 19. PubMed PMID: 18028317.

Latest revision as of 15:44, 16 October 2019


chbR N238S Y30C


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

ChbR is encoded within the chb operon itself. In the presence of chitobiose, ChbR binds to two direct repeats located between two NagC binding sites and activates the transcription of the chb operon by overlapping the -35 box of chbBp. In the absence of chitobiose, ChbR acts as a repressor of the chb operon. Monoacetylchitobiose-6-P has been experimentally determined to be the inducer for ChbR.

The sequence of chbR was obtained from E.coli K12 MG1655 via PCR.Then we performed inverse PCR to achieve the site-directed mutation of amino acids of chbR. According to the literature, we chose three site-directed mutation: chbRN238S

Source

ChbR was obtained from the genomic sequence of Escherichia coli str. K-12 substr.MG1655

References

[1]Plumbridge J, Pellegrini O. Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP. Mol Microbiol. 2004 Apr;52(2):437-49. PubMed PMID: 15066032.

[2]Kachroo AH, Kancherla AK, Singh NS, Varshney U, Mahadevan S. Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose. Mol Microbiol. 2007 Dec;66(6):1382-95. Epub 2007 Nov 19. PubMed PMID: 18028317.