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Category: Applied and Industrial Microbiology
Genetic Engineering Tools for Saccharomyces cerevisiae, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap20-1.gif /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap20-2.gifAbstract:
The yeast Saccharomyces cerevisiae is a widely used cell factory. Genetic engineering requires efficient transformation techniques, and many protocols for transformation of S. cerevisiae have been developed. These include methods involving spheroplast generation, electroporation, and biolistics with DNA coated microprojectiles. For expression of heterologous genes or overexpression of homologous genes in S. cerevisiae, plasmid vectors are usually employed. Most are shuttle vectors that allow cloning and amplification in Escherichia coli. Yeast plasmid vectors can be divided into different types. Probably the most widely used type of selectable markers are ones that confer prototrophy to auxotrophic strains containing mutations in amino acid and/or nucleotide biosynthetic pathways, e.g., LEU2 and URA3. Several of the prototrophic markers offer this possibility, the most widely used being the URA3 marker, which can be selected against using 5-fluoroorotic acid (5FOA). Proper transcription termination is a requirement for mRNA stability and thus also for high expression levels. Protein secretion requires the presence of an N-terminal signal sequence. Gene targeting is particularly efficient in S. cerevisiae because DNA double-strand breaks are preferentially repaired by homologous recombination as opposed to nonhomologous end joining. Since the codon composition at the gene’s 5’ end seems to be most important, it may be sufficient only to optimize the first 14 to 32 codons to achieve good expression.
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Genomic integration using an integrative plas-mid. YFG1 and a selective marker, in this case URA3, are cloned into an E. coli plasmid. The plasmid is linearized at a site within URA3 prior to transformation into a ura3 strain. After integration, the URA3 gene is duplicated, typically resulting in one functional and one nonfunctional copy. Chromosomal DNA is indicated by the presence of a gray oval representing the centromere.
Substrates for disruption and deletion of YFGl. (A) Disruption of YFG1 is achieved by using a linear substrate composed of a selectable marker (URA3) flanked by sequences up- and downstream of the integration site in YFG1. (B) Deletion of YFG1 is achieved by using a linear substrate composed of a selectable marker (URA3) flanked by sequences up- and downstream of YFG1. Original sequence between the YFG1 up- and downstream sequences will be deleted. (C) Construction of a gene deletion substrate by PCR. In a first round of PCR, fragments containing the up- and downstream sequences of YFG1 and the marker gene (URA3) are generated in three independent PCR reactions. Note that one of the primers in the primer pair used to generate the upstream fragment is extended by a sequence identical to the extreme 5′-upstream section of URA3 as indicated by a black line added to the gray primer. The sequence of the extension is complementary to the 5′-primer used to amplify URA3. Similarly, one of the primers used to amplify the downstream fragment is extended by a sequence that is complementary to the 3′-primer used to amplify URA3. The two primer extensions produce sequence overlaps that allow all three fragments to be fused and amplified in a second round of PCR. (D) Construction of a bipartite gene deletion substrate. The up- and downstream sections are generated as shown in panel C. In a first round of PCR, two fragments consisting of the 2/3 upstream and 2/3 downstream parts of URA3 are generated in two individual PCR reactions. The primer extensions added to primers used to generate the up- and downstream sequences allow the up- and downstream fragments to be individually fused to the 5′-2/3 and 3′-2/3 URA3 fragments in separate PCR reactions.
Substrates for iterative gene deletion. Direct repeats flanking the selectable marker allow the marker to be eliminated from the genome by recombination. If the selectable marker is URA3, recombinants can be selected using 5-FOA. (A) Construction of a bipartite gene deletion substrate with the marker flanked by a direct repeat (labeled DR). The template for the marker fragments is a plasmid that contains direct repeats flanking the marker. The remaining steps are similar to the legend for Fig. 2D . (B) Genomic integration of a bipartite gene-targeting substrate. Note that homologous recombination completes the selectable marker after cotransforming the two substrate fragments into the cell. (C) Direct repeat recombination eliminates the selectable marker from the genome. Note that one repeat remains in the genome. (D) By varying the sequence content of the DR repeats flanking the marker in the template plasmid, different useful sequences may be introduced between defined up and down sequences, as indicated; see text for details.
Incorporation of GFP DNA sequence into the genome. (A) Universal vector set for incorporating the GFP DNA sequence at any location in the genome using a bipartite gene-targeting substrate. Positions of the primers are indicated. (B) Strategy to extend the protein encoded by YFG1 C-terminally with GFP. The position of the primer pairs used to amplify the two targeting sequences is indicated. Each fragment will terminate with a sequence tag matching either the 5′ - or the 3′ -end of GFP as indicated. Note that a sequence complementary to a stop codon, shown as an open circle, must be incorporated in the forward primer used to amplify the downstream targeting fragment. This codon is positioned right after the GFP segment of this primer as indicated.
Substrates for introduction of point mutations. Identical direct repeats of YFG1 sequences containing the relevant mutation flank a selectable marker (URA3). The substrate is shown as a bipartite gene-targeting substrate. Direct repeat recombination between mutant repeats leaves only the mutation in the genome.
Marker genes
Promoters for heterologous gene expression
Available plasmid expression vector