gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
S. cerevisiae Gene Expression Vector
Our S. cerevisiae gene expression vector system is based on the widely used pYES2 vector. This is a powerful and efficient system for expressing recombinant proteins in yeast, or for studying gene function in yeast via overexpression. The gene of interest is cloned into the vector under the control of a promoter selected by the user. Several standard promoters are available for selection on VectorBuilder. One of them is the strong inducible promoter from the yeast galactokinase (GAL1) gene, which is the most commonly used promoter in yeast recombinant protein expression systems.
In typical yeast laboratory strains (e.g. INVSc1), the transcriptional activity of the GAL1 promoter is responsive to the carbon source present in the medium. The presence of glucose represses transcription from the GAL1 promoter, while galactose activates the promoter. Therefore, induction of the gene of interest can be achieved by simply removing the glucose-containing medium from the cells and replacing with galactose-containing medium.
Alternatively, raffinose may be used as a carbon source. Raffinose neither represses nor induces transcription from the GAL1 promoter, and addition of galactose is sufficient to activate the GAL1 promoter even in the presence of raffinose. Induction of the GAL1 promoter by galactose is more rapid in cells maintained in raffinose-containing medium when compared to cells maintained in glucose-containing medium. However, since raffinose does not repress the GAL1 promoter, this methodology can result in “leaky” expression of the gene of interest prior to induction.
In general, recombinant proteins can be detected in ~4h after induction with galactose in cells that have been maintained in glucose, and in ~2h in cells that have been cultured in raffinose. We recommend that you perform a time course to optimize expression of your recombinant protein.
For further information about this vector system, please refer to the papers below.
|Science. 127:28-9 (1958)
Mol. Cell. Biol. 4:1985-98 (1984)
Mol. Cell. Biol. 4:2467-78 (1984)
|The GAL1 promoter|
|Cell 40:767-774 (1985)||Induction of gene expression using the GAL1 promoter|
|Methods Enzymol. 194:1-863. (1991)||Extensive information about gene expression in yeast|
This vector system is designed for constitutive or inducible gene expression in S. cerevisiae. In the case of inducible expression, the gene of interest is cloned into the vector under the control of the GAL1 promoter, which allows induction of the gene of interest by addition of galactose to the medium. The presence of glucose in the medium will repress expression of the gene of interest. Raffinose can also be used as a carbon source that neither activates nor represses expression from the GAL1 promoter.
Strong expression: The inducible GAL1 promoter allows for very high-level expression of genes of interest.
Tightly controlled expression: Under the control of the GAL1 promoter, the expression of the gene of interest is generally very strongly repressed in the presence of glucose and strongly activated by galactose.
Rapid induction: Recombinant proteins expressed from the GAL1 promoter may be detectable in ~2 hours after induction in cells that have been cultured in raffinose.
Potential leaky expression: For protein expression driven by the GAL1 promoter, raffinose may be used as a carbon source instead of glucose or galactose. However, raffinose does not represses the GAL1 promoter, which can result in leaky expression of the gene of interest. Glucose needs to be used in the media for repression of the GAL1 promoter.
Key components of our vector
Promoter: The promoter that drives your gene of interest is placed here. When the inducible GAL1 promoter is used, galactose will induce high-level transcription of the gene of interest, while glucose will strongly repressed expression. Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
CYC1 terminator: Sequence which facilitates transcriptional termination and polyadenylation of mRNA in yeast.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
Marker: A yeast selectable marker is placed here. It allows the yeast cells successfully transformed with the vector to be selected. One commonly used marker is the orotidine-5'-phosphate decarboxylase (URA3) gene, which allows selection of yeast transformants in uracil or uridine deficient medium. Additionally, if 5-Fluoroorotic acid (5-FOA) is added to the media, the URA3 gene product will convert 5-FOA into 5-fluorouracil, which is a toxin that will cause cell death, thereby allowing selection against yeast carrying the plasmid.
2µ ori: Origin of replication which permits high-copy replication and maintenance in S. cerevisiae.