Difference between revisions of "Part:BBa K802004:Design"
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===Design Notes=== | ===Design Notes=== | ||
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==Original vector: pHT315== | ==Original vector: pHT315== | ||
| − | This shuttle vector is derived from pHT315. The complete sequence of pHT315 has been published, as well as the construction of | + | 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== | ==Construction of BBa_K802004== | ||
Latest revision as of 20:48, 26 September 2012
Shuttle vector for E. coli and B. subtilis
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 6506
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6506
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 6506
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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