A method for labeling proteins with tags at the native genomic loci in budding yeast

Abstract

Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique in the budding yeast Saccharomyces cerevisiae is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. However, several limitations hamper the application of this technique, such as the selectable markers not being reusable, tagging of only the C-terminal being possible, and a “scar” sequence being left in the genome. Here, we describe a strategy to solve these problems by tagging target genes based on a pop-in/pop-out and counter-selection system. Three fluorescent protein tag (mCherry, sfGFP, and mKikGR) and two epitope tag (HA and 3×FLAG) constructs were developed and utilized to tag HHT1, UBC13 or RAD5 at the chromosomal locus as proof-of-concept.

amdS as a dominant recyclable marker in Cryptococcus neoformans

“…In S. cerevisiae, the Blaster method has already been used not only to create fluorescent protein fusions, but also to add epitope tags (Wang et al, 2017). This approach could be expanded upon in C. neoformans by using our…”

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains

“…The ability to have a “marker-less” genetic alteration is often desirable, but can also present technical challenges. Prior to the introduction of CRISPR/Cas9, there have been other systems used to remove a selectable marker (often in at least two or more consecutive steps) using integration vectors (Sikorski and Hieter, 1989 ), the Cre-Lox system (Germino et al, 2006 ), or other methods, such as the classic “loop in-loop out” (Landgraf et al, 2016 ; Wang et al, 2017 ). This is often extremely useful in strains where the presence of multiple plasmids and multiple loci have all been (each) marked with the entire ensemble of selectable markers in yeast (or if a particular genetic background does not include the full suite of auxotrophic knockouts).…”

Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

“…Fluorescent proteins are a good tool for tagging target proteins in yeast, owing to the development of powerful genetic manipulation techniques in the past (Boeke et al, 1984 , 1987 ; Longtine et al, 1998 ; Wang et al, 2017 ). However, compared with organic dyes and Qdots, both the lower intensity and stability of fluorescent proteins restricts them for use in single-molecule studies.…”

“…pRS306/ RFA2-Gal-RFA3 and pRS303/ GAL4 - CBP-TEV-RFA1 are used for RPA expression (Yeeles et al, 2015 ), and pJF17, pJF18, and pJF19 are used for ORC expression (Frigola et al, 2013 ). To label RPA and ORC with Avi-tag, 3 × FLAG-AVI and AVI were added at the C-terminus of RFA2 in the plasmid pRS306/ RFA2-Gal-RFA3 and at the N-terminus of ORC1 before CBP in the plasmid pJF19 using overlap polymerase chain reaction (PCR) as described by Wang et al ( 2017 ) and subcloned; the two new plasmids were named pC-AVI and pN-AVI (Figures S1 , S2 ). pC-AVI was constructed with several detailed steps: (1) 3 × FLAG-AVI fragment was amplified from pFA6a- 3 × FLAG-AVI using FLAG-AVI-F/R primers, and RFA2 gene and a terminator were amplified from pRS306/ RFA2-Gal-RFA3 using RFA2-F/R and terminator-F/R primers, respectively; (2) RFA2-3 × FLAG-AVI was amplified by overlap PCR with RFA2-F/FLAG-AVI-R primers; (3) RFA2-3 × FLAG-AVI- terminator was amplified by overlap PCR with Final-F/R primers (Figure S1A ); and (4) the final fragment was inserted into Asc I- Xho I digested pRS306/ RFA2-Gal-RFA3 by homologous recombination using a quick-fusion cloning kit (B22611, Biotool).…”