Difference between revisions of "Part:BBa K2194000:Design"

 
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===Design Notes===
 
===Design Notes===
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[update]
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Primers used to PCR amplify <i>chrR</i> from the <i>Escherichia coli</i> MG1655 genome<sup>NOTE</sup>: <p>
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5’ <b>CGATCGTCTCACTCGA</b>ATGTCTGAAAAATTGCAGGTG 3’ </p><p>
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5'<b>GCATCGTCTCACTCTGCCA</b>TTAGATCTTAACTCGCTGAATAAACTC 3'</p>
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<br>
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Primers used for PCR site-directed mutagenesis (in addition to the primers used to amplify <i>chrR</i> from the genome<sup>NOTE</sup>:<p>
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5' <b>ATGCCGTCTCA<u>A</u></b>ATCACCTGCGCCAGATTC 3'</p><p>
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5' <b>ATGCCGTCTCAGAT<u>T</u>CTG</b>ACAGCGCGCGCCGCC 3'</p>
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<br>
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<p>The primers for PCR site-directed mutagenesis incorporate BsmBI recognition sites into the overhangs so that the two resulting PCR fragments can be assembled using a Golden Gate reaction. This process generates the sequence for <i>chrR6</i> without any scars around the site of PCR mutagenesis.</p>
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<br>
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NOTE: The primer overhangs are <b>bolded</b> and the part of the primer that introduces the mutation is <u>underlined</u>.
  
 
===Source===
 
===Source===
  
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Escherichia coli genome; mutation Tyr128Asn introduced with PCR site-directed mutagenesis.
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We sourced the nucleotide sequence for <i>chrR</i> from the <i>Escherichia coli</i> MG1655 genome NCBI database (entry NC_000913.3:3894652-3895218). We used this sequence to design primers to PCR amplify the sequence from purified <i>Escherichia coli</i> MG1655 genome. Then, we designed primers to incorporate a single nucleotide change so that the 128th codon of the sequence changed from TAT → AAT (Try → Asn).
  
 
===References===
 
===References===
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Nucleotide [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – . Accession No. NC_000913.3:3894652-3895218, chrR chromate reductase; [cited 2017 October 31]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3:3894652-3895218

Latest revision as of 02:09, 1 November 2017


chrR6 Chromate Reductase Enzyme


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 570
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 337
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3
    Illegal BsaI.rc site found at 583


Design Notes

Primers used to PCR amplify chrR from the Escherichia coli MG1655 genomeNOTE:

5’ CGATCGTCTCACTCGAATGTCTGAAAAATTGCAGGTG 3’

5'GCATCGTCTCACTCTGCCATTAGATCTTAACTCGCTGAATAAACTC 3'


Primers used for PCR site-directed mutagenesis (in addition to the primers used to amplify chrR from the genomeNOTE:

5' ATGCCGTCTCAAATCACCTGCGCCAGATTC 3'

5' ATGCCGTCTCAGATTCTGACAGCGCGCGCCGCC 3'


The primers for PCR site-directed mutagenesis incorporate BsmBI recognition sites into the overhangs so that the two resulting PCR fragments can be assembled using a Golden Gate reaction. This process generates the sequence for chrR6 without any scars around the site of PCR mutagenesis.


NOTE: The primer overhangs are bolded and the part of the primer that introduces the mutation is underlined.

Source

We sourced the nucleotide sequence for chrR from the Escherichia coli MG1655 genome NCBI database (entry NC_000913.3:3894652-3895218). We used this sequence to design primers to PCR amplify the sequence from purified Escherichia coli MG1655 genome. Then, we designed primers to incorporate a single nucleotide change so that the 128th codon of the sequence changed from TAT → AAT (Try → Asn).

References

Nucleotide [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – . Accession No. NC_000913.3:3894652-3895218, chrR chromate reductase; [cited 2017 October 31]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3:3894652-3895218