Difference between revisions of "Part:BBa K1172303:Design"

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we eliminated three illegal restriction sites: Therefore we substituted one base per illegal restriction site by using PCR. The designed PCR-primers generated homologue overlaps, thus enabling Gibson Assembly afterwards:
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we eliminated three illegal restriction sites: Therefore we substituted one base per 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)
 
* Pst1 at 1644 bp (changing CTGCA^G to CTGtAG)

Latest revision as of 22:03, 27 October 2013

Riboflavin synthesis gene cluster from shewanella oneidensis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1114
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

we isolated the rib-gene-cluster 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 rib-gene-cluster with pSB1C3. Insofar, we designed four primers to generate homologous overlaps between pSB1C3 and the rib-gene-cluster:

  • pSB1C3_fwd_Rib
    AGCTTGAACAACAGTTGTAATACTAGTAGCGGCCGCTGCAGTC
  • pSB1C3_rev_Rib
    TCGAGTACTGACCAATTCATCTCTAGAAGCGGCCGCGAATTCC
  • Rib_fwd
    GAATTCGCGGCCGCTTCTAGATGAATTGGTCAGTACTCGATAAC
  • Rib_rev
    CTGCAGCGGCCGCTACTAGTATTACAACTGTTGTTCAAGCTG


we eliminated three illegal restriction sites: Therefore we substituted one base per 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 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.