Abstract
Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5’ and 3’ untranslated region (UTR) and without introducing any extraneous unwanted “scar” sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells.
Highlights
The budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe) have both been extensively studied as model systems for eukaryotic cells
In S. pombe, large-scale protein localization studies were performed both by using 4910 open reading frames (ORFs)-YFP fusions integrated at the ectopic leu1 locus and driven by the inducible nmt1 promoter [6] or using strains with GFP endogenously fused to 1058 ORFs [7]
We describe two methods for endogenous gene tagging with fluorescent proteins in yeast: scarless C-terminal tagging (Fig 1B-i) and scarless N-terminal tagging (Fig 1B-ii)
Summary
The budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe) have both been extensively studied as model systems for eukaryotic cells These yeast cells are ideal model organisms for functional genomics and genome-wide gene engineering, partly because of their efficient homologous recombination machinery. One problem is that most of the C-terminal integration methods, beside a few exceptions [5,10,11], use an exogenous 3’ UTR, which disrupts any endogenous 3’ end mediated expression control of the tagged gene and can alter protein levels [12]. Any changes to the UTRs that may affect protein abundances are of concern, especially for quantitative studies that measure protein levels [3,18] These problems call for gene tagging methods that do not replace the endogenous UTRs or leave behind any unwanted DNA sequences in the tagged gene (i.e. a “scar”)
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