Part:BBa_K1172303:Design

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
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 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 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)