Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase (original) (raw)
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A shuttle vector series for precise genetic engineering ofSaccharomyces cerevisiae
Yeast, 2016
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.
Journal of Bacteriology, 1983
DNA sequences from the Candida utilis genome which, when cloned into a yeast integration plasmid (YIp5), confer on YIp5 the ability to replicate autonomously in Saccharomyces cerevisiae are described. Several recombinant plasmids which transform S. cerevisiae YNN27 to Ura3+ with an efficiency of 2 x 103 transformants per ,ug of DNA were obtained. One of the recombinant plasmids, pHMR22 (6.6 kilobases) contains ars (autonomous replication sequence), which is homologous with two different DNA fragments of the C. utilis genome but has no detectable homology to total DNA from Candida albicans, Pachysolen tannophilus, or S. cerevisiae. Restriction and subcloning analyses of pHMR22 showed that Sau3A destroys the functions of cloned ars whereas there are no BamHI, PstI, SalI, HindlIl, EcoRI, or PvuII sites in the region of ars which is required for its functional integrity. Thus, pHMR22 appears to be a useful vector for cloning desired genes in S. cerevisiae.
Targeted deletions created in yeast vectors by recombinational excision
Nucleic Acids Research, 1999
We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination. Two complementary single-stranded oligonucleotides are designed so that the 5′ and 3′ halves of the resulting double-stranded oligonucleotide are homologous to the 5′ and 3′ side of a desired deletion junction, respectively. The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast. By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide.
DNA insertion system for complex yeast shuttle vectors
Current Genetics, 1995
A DNA insertion system, termed marked homologous recombination, was devised for the construction of complex yeast shuttle plasmids. This system, which is efficient, rapid and easy to use, should contribute to our understanding of gene-gene interactions in yeast cells. Key words Saccharomyces cerivisiae Plasmid construction 9 Homologous recombination E. coli-yeast common markers It is relatively easy in vitro to insert DNA fragments into E. coti vectors such as the pUC (Yanish-Perron et al. 1985), the pBluescript (Stratagene) or the pGEM-Z (Promega) series of vectors. For this purpose, large polylinker regions are required, as well as a blue/white screening system involving [3-galactosidase activity and Xgal. However, when having to construct more complex yeast/E, coli shuttle plasmids which combine together several genes, this approach is more difficult because of the lack of adequate and unique restriction sites, a low construction yield, or problems in transforming E. coli with a very large construct. As the demand for complex shuttle plasmids is likely to grow in the future, new techniques for plasmid construction are required. A novel gene selection method in yeast, called the genegene interference (or GGI) method, was recently developed which requires the insertion of specific genes into a large general vector to give a reference plasmid used for subsequent specific GGI selections (Daniel 1993). For performing these constructions, it seemed convenient to first insert the specific genes into simple E. coli vectors, using blue/white screening, and then to transfer these genes to the complex yeast shuttle vector by utilizing yeast as the final executant taking advantage of its high propensity for
Gene, 1998
In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2 mm plasmid-derived ori and Flp/FRT machinery. To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100 kb apart. Moreover, 2 mm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites. The strain carrying all these inserted elements was perfectly stable. Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2 mm ori, as aided by FRT/Flp, provided the amplification function. Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.
Journal of Microbiological Methods, 2006
Increasing industrial competitiveness and productivity demand that recombinant yeast strains, used in many different processes, be constantly adapted and/or genetically improved to suit changing requirements. Among yeasts, Saccharomyces cerevisiae is the best-studied organism, and the most frequently employed yeast in industrial processes. In the present study, laboratory strains and industrial S. cerevisiae strains were stably transformed with a novel vector containing the glucoamylase cDNA of Aspergillus awamori flanked by δ-sequences (δGlucoδ), and lacking a positive selection marker. Co-transformation with known plasmids allowed selection by auxotrophic complementation of the leu2 mutation and/or geneticin resistance (G418 R ). In all cases, several copies of the δGlucoδ vector were inserted into the genome of the yeast cell without selective pressure, showing 100% stability after 80 generations. Transformation frequency of the new vector was similar for S. cerevisiae laboratory strains and industrial wild-type S. cerevisiae strains. This novel genetic transformation system is versatile and suitable to introduce several stable copies of a desired expression cassette into the genome of different S. cerevisiae yeast strains.
FEMS Yeast Research, 2014
Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log 10 mean AE 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
Metabolic engineering, 2015
Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achi...