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:
+
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 234 bp (changing CTGCA^G to CTGtAG)
 
* Pst1 at 234 bp (changing CTGCA^G to CTGtAG)
−
* Pst1 at 1644 bp (changing CTGCA^G to CTGtAG)
 
−
* EcoR1 at 1840 bp (changing G^AATTC to GAATTt)
 
  
−
These primers were used to eliminate the illegal restriction sites:
+
These primers were used to eliminate the illegal restriction site:
−
::*pst1-234 fwd (38 bp) <br> TGTCACCTTAGAACCCTGTAGCCATTATGGTCGTACGC
+
::*pst1 norM fwd <br> GCGATCGTGCTGATATTTCTGCGGTGGTCGCCAAAGTC
−
::*pst1-234 rev (32 bp)<br> CGACCATAATGGCTACAGGGTTCTAAGGTGAC
+
::*pst1 norM rev <br> CAATAAGCCGACTTTGGCGACCACCGCAGAAATATCAGCAC
−
::*pst1-1644 fwd (30 bp)<br> GTGCCCCATACTGTAGGTGAAACCACGTTG
+
 
−
::*pst1-1644 rev (34 bp)<br> AACGTGGTTTCACCTACAGTATGGGGCACAATCG
+
−
::*''Eco''R1 fwd (43 bp)<br> GGCACTCAGTTCACTTAGGTATAGAATTTATAACAACAGTCAC
+
−
::*''Eco''R1 rev (43 bp)<br> GTGACTGTTGTTATAAATTCTATACCTAAGTGAACTGAGTGCC
+
  
 
===Source===
 
===Source===

Revision as of 22:03, 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 234 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.