Difference between revisions of "Part:BBa K802004:Design"

 
 
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===Design Notes===
 
===Design Notes===
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The SpeI site from the B. subtilis coding region of the pHT315 plasmid was eliminated by filling-in. Afterwards, an iGEM linker containing all 4 of the restriction enzyme sites (EcoRI,XbaI,SpeI,PstI) was cloned into the modified vector.
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==Original vector: pHT315==
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This shuttle vector is derived from pHT315. The complete sequence of pHT315 has been published, as well as the construction of pHT315  in "Construction of cloning vectors for Bacillus thuringiensis (Arantes O. & Lereclus D., 1991 Gene[1]"). The E. coli origin comes from pUC19 and the Bacillus origin from pHT1035. The newly constructed plasmid was modified so that the final construct (pHT315) would not have any restriction site duplicated. 
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==Construction of BBa_K802004==
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The pHT304 shuttle vector contains all 4 of the iGEM sites. The gel electrophoresis showed that all of them are unique sites. Given the fact that the polylinker from pHT315 is the same one from pUC19, we know that the sites EcoRI, XbaI and PstI are in the polylinker and not in a coding region of the plasmid. However, the SpeI site was not in the polylinker. A double digestion by EcoRI and SpeI gave two fragments of 1 kb and 5.5 kb. At a closer look at the Bacillus region coming from the pHT1035-15△HindIII plasmid (i.e. the pHT1035 after the deletion of the site HindIII and after the selection of a high copy number plasmid based on the antibiotic concentration), we identified the SpeI site. Fortunately, the site was neither in the origin of replication for Bacillus, nor in the gene responsible for the Erythromycin resistance.
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The site SpeI was eliminated by filling-in [2). Afterwards, a customized iGEM polylinker [3] containing all 4 of the iGEM sites in the right order was cloned in EcoRI and HindII sites of the shuttle vector.
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===References===
 
===References===
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[1] http://download.bioon.com.cn/upload/month_0904/20090402_5d481d87240fe39e12cb3TdyPxwuDKxP.attach.pdf           
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[2] https://static.igem.org/mediawiki/2012/1/13/Filling-in_Protocol.pdf                                                 
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[3] https://parts.igem.org/Part:BBa_K802005

Latest revision as of 20:48, 26 September 2012

Shuttle vector for E. coli and B. subtilis


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 6506
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6506
    Illegal NheI site found at 3377
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 6512
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6506
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 6506
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 6506
    Illegal XbaI site found at 6521
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3203
    Illegal BsaI.rc site found at 5190


Design Notes

Original vector: pHT315

This shuttle vector is derived from pHT315. The complete sequence of pHT315 has been published, as well as the construction of pHT315 in "Construction of cloning vectors for Bacillus thuringiensis (Arantes O. & Lereclus D., 1991 Gene[1]"). The E. coli origin comes from pUC19 and the Bacillus origin from pHT1035. The newly constructed plasmid was modified so that the final construct (pHT315) would not have any restriction site duplicated.

Construction of BBa_K802004

The pHT304 shuttle vector contains all 4 of the iGEM sites. The gel electrophoresis showed that all of them are unique sites. Given the fact that the polylinker from pHT315 is the same one from pUC19, we know that the sites EcoRI, XbaI and PstI are in the polylinker and not in a coding region of the plasmid. However, the SpeI site was not in the polylinker. A double digestion by EcoRI and SpeI gave two fragments of 1 kb and 5.5 kb. At a closer look at the Bacillus region coming from the pHT1035-15△HindIII plasmid (i.e. the pHT1035 after the deletion of the site HindIII and after the selection of a high copy number plasmid based on the antibiotic concentration), we identified the SpeI site. Fortunately, the site was neither in the origin of replication for Bacillus, nor in the gene responsible for the Erythromycin resistance. The site SpeI was eliminated by filling-in [2). Afterwards, a customized iGEM polylinker [3] containing all 4 of the iGEM sites in the right order was cloned in EcoRI and HindII sites of the shuttle vector.



Source

The plasmid is derived from the pHT315 vector (Arantes and Lereclus, 1991).

References

[1] http://download.bioon.com.cn/upload/month_0904/20090402_5d481d87240fe39e12cb3TdyPxwuDKxP.attach.pdf [2] https://static.igem.org/mediawiki/2012/1/13/Filling-in_Protocol.pdf [3] https://parts.igem.org/Part:BBa_K802005