title: A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae.
abstract: Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single- and multi-copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double-crossover recombination mechanism, resulting in stable single- and multi-copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control.
authors.affiliation: D-BSSE, ETH Zurich and Swiss Institute of Bioinformatics, Zurich, Switzerland.|Life Science Zurich PhD Programme on Molecular and Translational Biomedicine, Zurich, Switzerland.|Competence Centre for Personalized Medicine, Zurich, Switzerland.
authors.forename: Robert
authors.initials: R
authors.lastname: Gnügge
authors.affiliation: D-BSSE, ETH Zurich and Swiss Institute of Bioinformatics, Zurich, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Liphardt
authors.affiliation: D-BSSE, ETH Zurich and Swiss Institute of Bioinformatics, Zurich, Switzerland.
authors.forename: Fabian
authors.identifier: http://orcid.org/0000-0001-5264-333X
authors.initials: F
authors.lastname: Rudolf
mesh_terms: D002503:Centromere / Q000235:genetics; D003001:Cloning, Molecular; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
keywords: Saccharomyces cerevisiae; gene dosage; genomic integration; plasmid loss; plasmids; shuttle vectors