N‐ICE plasmids for generating N‐terminal 3 × FLAG tagged genes that allow inducible, constitutive or endogenous expression in Saccharomyces cerevisiae

Abstract

PCR-mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N-terminus or the C-terminus of targeted genes. Although strategies of C-terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C-terminal epitope tagging is not practical for all proteins. For example, a C-terminal tag may affect protein function or a protein may get cleaved or processed, resulting in the loss of the epitope tag. Therefore, N-terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N-terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre-LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N-terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N-terminal tagging, which combines two previous strategies for epitope tagging in a haploid strain. These 'N-ICE' plasmids were constructed so that non-essential and essential genes can be N-terminally 3 × FLAG tagged and expressed from an inducible promoter (GAL1), constitutive promoters (CYC1 or PYK1) or the endogenous promoter. We have validated the N-ICE plasmid system by N-terminal tagging two non-essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1). Copyright © 2016 John Wiley & Sons, Ltd.

A Novel Sterol-Signaling Pathway Governs Azole Antifungal Drug Resistance and Hypoxic Gene Repression in Saccharomyces cerevisiae

“…The yeast plasmids used in this study were constructed and PCR verified as previously described (South et al 2010(South et al , 2013Mersman et al 2012;Zhang et al 2016). FLAG-tagged strains were generated using the N-ICE plasmid tagging system and PCR verified (Zhang et al 2016).…”

“…Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA) per the manufacturer's instructions. Primer Express 3.0 software was used for designing primers (see Table S3 in File S7) and quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described (South et al 2013;Zhang et al 2016). Three biological replicates, including three technical replicates, were performed for all samples.…”

“…Because a set4D strain is resistant to ketoconazole and fluconazole, we hypothesized that overexpression of Set4 would lead to azole hypersensitivity. To test this hypothesis, we integrated a constitutive PYK1 promoter upstream of SET4 and SET3 using our N-ICE plasmid system and performed a dilution assay on ketoconazole and fluconazole plates (Zhang et al 2016). Overexpression of Set4 resulted in hypersensitive growth compared to WT under ketoconazole or fluconazole treatment, suggesting that too much Set4 negatively affects cell viability ( Figure 1G).…”

Direct PA-binding by Chm7 is required for nuclear envelope surveillance at herniations

“…All yeast strains used in this study are listed in Table S1. Genetic modifications to generate gene deletions or the in-frame incorporation of fluorescent protein genes was accomplished using a PCR-based approach using the pFA6a plasmid series or pK3F (Table S2) as templates (Longtine et al, 1998; Zhang et al, 2017). Further, to generate DTCPL2030 loxP/Cre-mediated gene disruption was used to delete the coding sequence for the hydrophobic stretch (H) within the endogenous CHM7 gene.…”

“…Further, to generate DTCPL2030 loxP/Cre-mediated gene disruption was used to delete the coding sequence for the hydrophobic stretch (H) within the endogenous CHM7 gene. Briefly, a kanamycin resistance cassette ( kan-MX6 ) flanked by loxp sites was amplified off of pK3F (Zhang et al, 2017) using primers with 60 bp homology arms that flank the region targeted for deletion. The PCR product was transformed into DTCPL515 (in this strain the CHM7 locus is modified to express chm7-N-GFP behind the GAL1 promoter) and kanamycin-resistant colonies were selected.…”