Difference between revisions of "Part:BBa K1172302:Design"
 
Line 6: Line 6:
  
 
===Design Notes===
 
===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:
+
we isolated the ''ribC'' gene 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 ''ribC'' with pSB1C3. Insofar, we designed four primers to generate homologous overlaps between pSB1C3 and the ''ribC'' gene:
−
::*pSB1C3_fwd_Rib <br> AGCTTGAACAACAGTTGTAATACTAGTAGCGGCCGCTGCAGTC
+
::*pSB1C3_fwd_RibC <br> GTTTTAACGGACTATCTTAATACTAGTAGCGGCCGCTGCAGTCC
−
::*pSB1C3_rev_Rib <br> TCGAGTACTGACCAATTCATCTCTAGAAGCGGCCGCGAATTCC
+
::*pSB1C3_rev_RibC <br> TGAACTATACCAGTAAACATCTCTAGAAGCGGCCGCGAATTCC
−
::*Rib_fwd <br> GAATTCGCGGCCGCTTCTAGATGAATTGGTCAGTACTCGATAAC
+
::*RibC_fwd <br> GAATTCGCGGCCGCTTCTAGATGTTTACTGGTATAGTTCAAGC
−
::*Rib_rev <br> CTGCAGCGGCCGCTACTAGTATTACAACTGTTGTTCAAGCTG
+
::*RibC_rev <br> CTGCAGCGGCCGCTACTAGTATTAAGATAGTCCGTTAAAACG
  
  
−
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) <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===

Latest revision as of 22:28, 27 October 2013

ribC: 6,7-dimethyl-8-ribityllumazine synthase alpha subunit RibC


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 196


Design Notes

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

  • pSB1C3_fwd_RibC
    GTTTTAACGGACTATCTTAATACTAGTAGCGGCCGCTGCAGTCC
  • pSB1C3_rev_RibC
    TGAACTATACCAGTAAACATCTCTAGAAGCGGCCGCGAATTCC
  • RibC_fwd
    GAATTCGCGGCCGCTTCTAGATGTTTACTGGTATAGTTCAAGC
  • RibC_rev
    CTGCAGCGGCCGCTACTAGTATTAAGATAGTCCGTTAAAACG


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.