Difference between revisions of "Part:BBa K863204"
JSchirmacher (Talk | contribs) |
JSchirmacher (Talk | contribs) |
||
| Line 6: | Line 6: | ||
The shuttle vector consists of the plasmid pSB1C3, 5' UTR of alcohol oxidase 1 gene (''aox1'') containing the ''aox1'' promoter region, Kozak sequence, mating factor alpha 1 (MFalpha1), ''Aar''I restriction site, ''aox1'' terminator, ''his4'' gene and 3' UTR of ''aox1'' gene. Cloning and plasmid replication in ''E. coli'' are able via the pSB1C3 part. The gene of interest (like laccase) can be included in frame with MFalpha1 via AarI restriction site. With the N-terminal MFalpha1 the POI could be secreted in the media. Genomic integation of MFalpha1-taged POI is able via the 5' UTR and 3' UTR of the ''aox1'' gene. This allows a double cross over and the genomic integration without any bacterial proportion of DNA which could be a decisive point for industrial application. The complementation of histidine auxotrophie via ''his4'' gene was chosen instead of a zeocine resistance. This selection strategy is chosen because we want to avoid the application of antibiotics. | The shuttle vector consists of the plasmid pSB1C3, 5' UTR of alcohol oxidase 1 gene (''aox1'') containing the ''aox1'' promoter region, Kozak sequence, mating factor alpha 1 (MFalpha1), ''Aar''I restriction site, ''aox1'' terminator, ''his4'' gene and 3' UTR of ''aox1'' gene. Cloning and plasmid replication in ''E. coli'' are able via the pSB1C3 part. The gene of interest (like laccase) can be included in frame with MFalpha1 via AarI restriction site. With the N-terminal MFalpha1 the POI could be secreted in the media. Genomic integation of MFalpha1-taged POI is able via the 5' UTR and 3' UTR of the ''aox1'' gene. This allows a double cross over and the genomic integration without any bacterial proportion of DNA which could be a decisive point for industrial application. The complementation of histidine auxotrophie via ''his4'' gene was chosen instead of a zeocine resistance. This selection strategy is chosen because we want to avoid the application of antibiotics. | ||
| − | Gibson assembly was used for building the shuttle vector | + | Gibson assembly was used for building the shuttle vector and the fragments with 5' overlap were amplified via PCR. In addition, the fragments were designed as basic BioBrick parts for different applications by the community. |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 03:01, 27 September 2012
shuttle vector pECPP11JS for recombination of genes of interest in yeast
The design of a minimal shuttle vector system with defined regions (or better DNA fragments) for expression and secretion of proteins of interest (POI), like laccase, which needed glycolisation is the basic concept. The shuttle vector needs a bacterial part for cloning in bacteria (like E. coli) and an eucaryotic part for genomic integration and selection in yeast (like P. pastoris). An other team will be able to clime in frame their gene of interest via the AarI restriction site. With only one restriction ligation cloning step the shuttle vector will be ready to use and integrate in the eucaryote P. pastoris.
The shuttle vector consists of the plasmid pSB1C3, 5' UTR of alcohol oxidase 1 gene (aox1) containing the aox1 promoter region, Kozak sequence, mating factor alpha 1 (MFalpha1), AarI restriction site, aox1 terminator, his4 gene and 3' UTR of aox1 gene. Cloning and plasmid replication in E. coli are able via the pSB1C3 part. The gene of interest (like laccase) can be included in frame with MFalpha1 via AarI restriction site. With the N-terminal MFalpha1 the POI could be secreted in the media. Genomic integation of MFalpha1-taged POI is able via the 5' UTR and 3' UTR of the aox1 gene. This allows a double cross over and the genomic integration without any bacterial proportion of DNA which could be a decisive point for industrial application. The complementation of histidine auxotrophie via his4 gene was chosen instead of a zeocine resistance. This selection strategy is chosen because we want to avoid the application of antibiotics.
Gibson assembly was used for building the shuttle vector and the fragments with 5' overlap were amplified via PCR. In addition, the fragments were designed as basic BioBrick parts for different applications by the community.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 4086
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3381
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1
Illegal BglII site found at 1532
Illegal BamHI site found at 1889
Illegal XhoI site found at 1196 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 4086
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 4086
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4242
Illegal BsaI.rc site found at 2041