Coding

Part:BBa_K1172301:Design

Designed by: Thorben Meyer   Group: iGEM13_Bielefeld-Germany   (2013-09-19)
Revision as of 22:00, 27 October 2013 by ThorbenM (Talk | contribs)

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.