Difference between revisions of "Part:BBa K1172301:Design"
 
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we eliminated one illegal restriction site: Therefore we substituted one base in the illegal restriction site by using PCR. The designed PCR-primers generated homologue overlaps, thus enabling Gibson Assembly afterwards:
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we eliminated one illegal restriction site: Therefore we substituted one base in the illegal restriction site by using PCR. The designed PCR-primers generated homologous overlaps, thus enabling Gibson Assembly afterwards:
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* Pst1 at 234 bp (changing CTGCA^G to CTGtAG)
+
 
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* Pst1 at 1644 bp (changing CTGCA^G to CTGtAG)
+
* Pst1 at 929 bp (changing CTGCA^G to CTGtAG)
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* EcoR1 at 1840 bp (changing G^AATTC to GAATTt)
+
 
 +
These primers were used to eliminate the illegal restriction site:
 +
::*pst1 norM fwd <br> GCGATCGTGCTGATATTTCTGCGGTGGTCGCCAAAGTC
 +
::*pst1 norM rev <br> CAATAAGCCGACTTTGGCGACCACCGCAGAAATATCAGCAC
  
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These primers were used to eliminate the illegal restriction sites:
 
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::*pst1-234 fwd (38 bp) <br> TGTCACCTTAGAACCCTGTAGCCATTATGGTCGTACGC
 
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::*pst1-234 rev (32 bp)<br> CGACCATAATGGCTACAGGGTTCTAAGGTGAC
 
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::*pst1-1644 fwd (30 bp)<br> GTGCCCCATACTGTAGGTGAAACCACGTTG
 
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::*pst1-1644 rev (34 bp)<br> AACGTGGTTTCACCTACAGTATGGGGCACAATCG
 
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::*''Eco''R1 fwd (43 bp)<br> GGCACTCAGTTCACTTAGGTATAGAATTTATAACAACAGTCAC
 
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::*''Eco''R1 rev (43 bp)<br> GTGACTGTTGTTATAAATTCTATACCTAAGTGAACTGAGTGCC
 
  
 
===Source===
 
===Source===

Latest revision as of 22:08, 27 October 2013

norM: Na+ Antiporter


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

we isolated norM from the genomic sequence of shewanella oneidensis MR-1. We designed our primers in a way that enabled us to use Gibson Assembly to ligate the norM gene with pSB1C3. Insofar, we designed four primers to generate homologous overlaps between pSB1C3 and norM:

  • norM_fwd
    GAATTCGCGGCCGCTTCTAGATGAAACATTCAGGCTTTCAAGCCAAAC
  • norM_rev
    CTGCAGCGGCCGCTACTAGTATTAATGGTGCACGCTGTCGCCTTC
  • norM_fwd
    GAATTCGCGGCCGCTTCTAGATGAAACATTCAGGCTTTCAAGCCAAAC
  • norM_rev
    CTGCAGCGGCCGCTACTAGTATTAATGGTGCACGCTGTCGCCTTC


we eliminated one illegal restriction site: Therefore we substituted one base in the illegal restriction site by using PCR. The designed PCR-primers generated homologous overlaps, thus enabling Gibson Assembly afterwards:

  • Pst1 at 929 bp (changing CTGCA^G to CTGtAG)

These primers were used to eliminate the illegal restriction site:

  • pst1 norM fwd
    GCGATCGTGCTGATATTTCTGCGGTGGTCGCCAAAGTC
  • pst1 norM rev
    CAATAAGCCGACTTTGGCGACCACCGCAGAAATATCAGCAC


Source

Shewanella oneidensis MR-1 genomic sequence.

  • The strain was kindly provided by Dr. Johannes Gescher (Karlsruher Institut für Technologie)

References

Schicklberger M, Sturm G, Gescher J: Genomic Plasticity Enables a Secondary Electron Transport Pathway in Shewanella oneidensis. Appl Environ Microbiol., 2013 Feb; vol. 79 no. 4 1150-1159.