Difference between revisions of "Part:BBa K863204"

Revision as of 02:58, 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 (see the figure below) 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. The origin of the DNA sequence for design of the shuttle vector and the source of DNA for PCR is listed in the table below.

Elements of the shuttle vector and their origin

Element	BioBrick	Origin of DNA sequence of design	Origin of DNA sequence of PCR
pSB1C3	pSB1C3	pSB1C3	pSB1C3
5'UTR of aox1	K863200	[http://products.invitrogen.com/ivgn/product/V19520 plasmid pPICZalphaA (Invitrogen)]	P. pastoris wild type X-33
Kozak sequence	J63003	BBa_J63003	integrated in primer sequence
MFalpha1	K863206	[http://products.invitrogen.com/ivgn/product/V19520 plasmid pPICZalphaA (Invitrogen)]	[http://products.invitrogen.com/ivgn/product/V19520 plasmid pPICZalphaA (Invitrogen)]
aox1 terminater	K863203	plasmid pPIC9K	P. pastoris wild type X-33
his4	K863202	plasmid pPIC9K	P. pastoris wild type X-33
3'UTR of aox1	K863201	plasmid pPIC9K	P. pastoris wild type X-33

Plasmid map for shuttle vector pSB1C3::BBa_K863204.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 4086
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 3381
21
INCOMPATIBLE 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
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 4086
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 4086
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 4242
Illegal BsaI.rc site found at 2041

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K863204 short</partinfo>
 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 ''Aar''I restriction site. With only one restriction ligation cloning step the shuttle vector will be ready to use and integrate in the eucaryote ''P. pastoris''.