Difference between revisions of "Part:BBa K1172301:Design"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1172301 short</partinfo>
 
<partinfo>BBa_K1172301 short</partinfo>
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===Design Notes===
 
===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 <br> GAATTCGCGGCCGCTTCTAGATGAAACATTCAGGCTTTCAAGCCAAAC
 +
::*norM_rev <br> CTGCAGCGGCCGCTACTAGTATTAATGGTGCACGCTGTCGCCTTC
 +
::*norM_fwd <br> GAATTCGCGGCCGCTTCTAGATGAAACATTCAGGCTTTCAAGCCAAAC
 +
::*norM_rev <br> 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 homologue overlaps, thus enabling Gibson Assembly afterwards:
 +
* 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:
 +
::*pst1-234 fwd (38 bp) <br> TGTCACCTTAGAACCCTGTAGCCATTATGGTCGTACGC
 +
::*pst1-234 rev (32 bp)<br> CGACCATAATGGCTACAGGGTTCTAAGGTGAC
 +
::*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===
  
−
 
+
''Shewanella oneidensis'' MR-1 genomic sequence.
−
 
+
*The strain was kindly provided by Dr. Johannes Gescher (Karlsruher Institut für Technologie)
  
 
===References===
 
===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.

Revision as of 22:00, 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 homologue overlaps, thus enabling Gibson Assembly afterwards:

  • 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:

  • pst1-234 fwd (38 bp)
    TGTCACCTTAGAACCCTGTAGCCATTATGGTCGTACGC
  • pst1-234 rev (32 bp)
    CGACCATAATGGCTACAGGGTTCTAAGGTGAC
  • pst1-1644 fwd (30 bp)
    GTGCCCCATACTGTAGGTGAAACCACGTTG
  • pst1-1644 rev (34 bp)
    AACGTGGTTTCACCTACAGTATGGGGCACAATCG
  • EcoR1 fwd (43 bp)
    GGCACTCAGTTCACTTAGGTATAGAATTTATAACAACAGTCAC
  • EcoR1 rev (43 bp)
    GTGACTGTTGTTATAAATTCTATACCTAAGTGAACTGAGTGCC

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.