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Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae

Johansson, Björn and Hahn-Hägerdal, Bärbel LU (2004) In Recombinant Gene Expression Reviews and Protocols ( Methods in Molecular Biology ; 267 )
Abstract
Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK... (More)
Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2µ-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001. (Less)
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author
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
in
Recombinant Gene Expression Reviews and Protocols ( Methods in Molecular Biology ; 267 )
editor
Balbas, Paulina and Lorence, Argelia
publisher
Humana Press
external identifiers
  • Scopus:4644343801
ISBN
1-59259-774-2
language
English
LU publication?
yes
id
8377be6b-1139-492a-8322-9fc96963baf9 (old id 529961)
alternative location
http://dx.doi.org/10.1226/1592597742
date added to LUP
2007-09-26 09:41:01
date last changed
2017-02-19 04:29:21
@inbook{8377be6b-1139-492a-8322-9fc96963baf9,
  abstract     = {Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2µ-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001.},
  author       = {Johansson, Björn and Hahn-Hägerdal, Bärbel},
  editor       = {Balbas, Paulina and Lorence, Argelia},
  isbn         = {1-59259-774-2},
  language     = {eng},
  publisher    = {Humana Press},
  series       = {Recombinant Gene Expression Reviews and Protocols ( Methods in Molecular Biology ; 267 )},
  title        = {Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae},
  year         = {2004},
}