Yeast Cloning Vectors and Genes

Lundblad, Victoria

doi:10.1002/0471142727.mb1304s21

Cited by 7 publications

(11 citation statements)

References 22 publications

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“…Yeast shuttle vectors allow for cloning and manipulation of DNA sequences in Escherichia coli and for the direct transfer and analysis of their effects in Saccharomyces cerevisiae (Lundblad, ). To propagate them in yeast, these plasmids bear sequences responsible for genomic integration or extrachromosomal maintenance and a selectable marker.…”

Section: Introductionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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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.

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Section: Introductionmentioning

confidence: 99%

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Gnügge

Liphardt

Rudolf

2016

Yeast

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“…Yeast HR assembly relies on shuttle vectors, plasmids that have replication origins and selectable markers that function in S. cerevisiae and in E. coli (Sikorski and Hieter, , and Table in UNIT here, Lundblad, ). Since it is unlikely that a preferred expression vector will be a yeast shuttle vector, the investigator needs to add these elements to their preferred plasmid backbone, either before assembly or as additional elements in a single multisegment assembly.…”

Section: Resultsmentioning

confidence: 99%

Overview of Post Cohen‐Boyer Methods for Single Segment Cloning and for Multisegment DNA Assembly

Sands

Brent

2016

CP Molecular Biology

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In 1973, Cohen and coworkers published a foundational paper describing the cloning of DNA fragments into plasmid vectors. In it, they used DNA segments made by digestion with restriction enzymes and joined these in vitro with DNA ligase. These methods established working recombinant DNA technology and enabled the immediate start of the biotechnology industry. Since then, “classical” recombinant DNA technology using restriction enzymes and DNA ligase has matured. At the same time, researchers have developed numerous ways to generate large, complex, multisegment DNA constructions that offer advantages over classical techniques. Here, we provide an overview of “post-Cohen-Boyer” techniques used for cloning single segments into vectors (T/A, Topo cloning, Gateway and Recombineering) and for multisegment DNA assembly (Biobricks, Golden Gate, Gibson, Yeast homologous recombination in vivo, and Ligase Cycling Reaction). We compare and contrast these methods and also discuss issues that researchers should consider before choosing a particular multisegment DNA assembly method.

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“…This interspecific complementation method might have succeeded because of the use of high-copy number plasmid vectors, which allowed sufficient expression of yeast genes in E. coli. S. cerevisiae genes were cloned by the complementation of S. cerevisiae mutants with S. cerevisiae plasmid libraries (Beggs, 1978;Hinnen et al, 1978;Hsiao and Carbon, 1979) and later genomic DNA libraries consisting of DNA fragments randomly inserted into yeast vectors using E. coli as a cloning host have been used for complementation cloning (Lundblad, 2001;Rose and Broach, 1991). Therefore, the cloning of genes into plasmid vectors was a common tool for gene manipulations in yeast as well as other species (Bergkamp et al, 1993;Janssen and Chen, 1998).…”

Section: Identification Of K Marxianus Auxotrophic Mutants With Genementioning

confidence: 99%

“…In addition to the integrative transformation of yeast with linear PCR-amplified DNA fragments, the utilization of S. cerevisiae genes for the complementation of K. marxianus auxotrophic mutants is also a novel strategy. Generally, heterologous promoters show lower expression than the native host promoter; therefore, promoter exchange or utilization of a multicopy vector is routinely used to express genes in heterologous host species (Choo et al, 1986;Kudla et al, 1988;Lundblad, 2001). In this study, however, we utilized PCRamplified S. cerevisiae genes for the direct and efficient interspecific complementation of K. marxianus mutants without promoter exchange or plasmid construction.…”

Section: Identification Of K Marxianus Auxotrophic Mutants With Genementioning

confidence: 99%

“…In addition to useful host strains, various host-vector systems consisting of yeast-Escherichia coli shuttle vectors have been constructed. For example, yeast YCp-type shuttle plasmids contain an autonomously replicating sequence and a centromere for stable plasmid maintenance, and YEp shuttle plasmids contain the 2μ origin for multicopy amplification in yeast cells (Lundblad, 2001). Using these host-vector systems and the gene transformation technique, many evolutionally conserved genes were identified from genetic mutants of S. cerevisiae by the complementation cloning.…”

Section: Introductionmentioning

confidence: 99%

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Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yarimizu

Nonklang

Nakamura

et al. 2013

Yeast

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The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

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Yeast Cloning Vectors and Genes

Cited by 7 publications

References 22 publications

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Overview of Post Cohen‐Boyer Methods for Single Segment Cloning and for Multisegment DNA Assembly

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

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