Construction of a novel plasmid system for epitope tagging and gene deletion in Saccharomyces cerevisiae

Abstract:

[Objective] For yeast gene functional studies, we constructed a plasmid set combining the advantages of pUG and pFA6a plasmid series, and allowing convenient insertion of tandem epitope tags using isocaudomers. [Methods] We cloned the loxP locus of pUG plasmids, the multiple restriction site of pFA6a plasmids and ADH1 terminator cassette by PCR. Through homologous recombination of the DNA fragments, we obtained plasmid pCLHN-TRP and pCLHN-URA. Using introduced isocaudomer sites, we then constructed additional tagging plasmids containing either single or multiple tandem copies of various epitope tags. Finally, we tested the performance of our plasmids using ATG1, COX4 and NHX1 as target genes. [Results] We constructed two plasmids for gene deletion purposes, and seventeen plasmids for epitope tagging purposes (covering 1-8 FLAG, 1-12 V5, 3-9 HA, 2-8 MYC, GFP and mCherry). Testing on selected target genes demonstrated the usability of these plasmids. In particular, by combining different copy numbers of tandem epitopes, it was feasible to properly detect proteins with drastically different expression levels on the same blot without saturating the signal of high expression targets. [Conclusion] The pCLHN plasmids constitute a beneficial complement to existing yeast plasmid tools.