Tom Drake1, Maya Schuldiner2
1Singer Instruments, Roadwater, Somerset, UK
2Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
A systematic collection of strains, or library, is an increasingly beneficial tool for biological research. Libraries can be used to study a broad unknown, utilising a knockout library to form the basic understanding of a principle, or they can be used to investigate a specific question over the whole genome. Manipulating these libraries used to be a labour intensive and time consuming process. Although the research potential often outweighed this cost, it could still be hard to justify the expenditure. Developments to the methodology of creating libraries have been crucial to increasing their use and acceptance as a vital tool in modern research. One such development was the incorporation of a specific gene tag, offering the ability to swap these tags to easily create a new library of interest. Developed by Anton Khmelinskii, Matthias Meurer (Michael Knop Laboratory, University of Heidelberg, Germany)1, this gene tagging process was furthered by collaboration with Ido Yofe and Uri Weill (Maya Schuldiner, Weizmann Institute of Science, Israel)2. They collectively developed the SWAp-Tag protocol to allow for quick and easy library customisation, from a parental collection. SWAp-Tag libraries have already been used to study a variety of topics within S. cerevisiae, and demonstrated their effectiveness over traditional library creation.
Creating a complete library of tagged S. cerevisiae is not a small task, and given that to completely investigate a topic, there’s a high chance that one library is not enough; the workload can quickly increase. Imagine if there was a tagged library with ~90% coverage of the yeast genome, in which you were also able to quickly swap the tag to meet your requirements3. This is exactly what Weil and Yofe et al (N’ library) and Meurer et al (C’ library) have recently shown in Nature Methods4, 5.
Utilising a technique termed SWAp-Tag (SWAT), a known tag was added to the N-terminus4 or C-terminus5 of each yeast protein3. This tag is hugely beneficial as it contains two known linker sequences at either end; L1 and L2. Utilising this, a designed plasmid carrying linker sequences and a selection marker can be cloned in – or swapped. As the linker sequences are always present, these can be used as primers for PCR to confirm the presence of the desired tag. This gives vast flexibility to the protocol as any tag of choice can be inserted, and verified through simple procedures. An overview of the SWAp-Tag (SWAT) protocol is shown in Figure 1.
Figure 1: Overview of the SWAp-Tag (SWAT) technology to create yeast libraries. b) Using an acceptor library of SWAT tagged genes, creating a new library is easy. Through crossing a donor library, which encodes a galactose induced I-SceI, and harboring a donor plasmid with the linker sequences (L1/L2). Two systematic restriction sites guide donor insertion. Through mating, haploids containing all genetic material are generated, and I-SceI expression is induced through growth on galactose, leading to double strand breaks in both the acceptor and donor, which is repaired through homolgous recombination. Correct swapping is then selected for in the new library. c) Donor plasmid features, which result in several types of tagged-gene libraries. This can be performed on N′, or C′ tagged acceptor libraries. Figure adapted from Yofe and Weil, et al. (2016)2.
In a recent development to the SWAT library, Weill and Yofe et al. also created a library tagging the N-terminus of ~90% of yeast genes4. They went on to create several different libraries from this parental strain to show the versatility of the library and demonstrate how beneficial this methodology can be in current research.
“Creation of new libraries would not have been possible without our Singer ROTOR robot”.
Prof. Maya Schuldiner – 2017
With the ability to easily swap the tag in the SWAT library, Weill and Yofe et al. modified the parental library in several key ways, which are outlined in Figure 2.
To examine the role of promoters, the parental library, which contains a NOP1 promoter, was reverted to the native promoter (and localisation sequence) whilst including GFP. A secondary library was also created, utilising TEF2, one of the strongest promoters known in yeast, and including mCherry. This was two fold in the benefits, the difference in promoter strength could be investigated, and this could be visualised with a different fluorophore. These libraries gave an insight into the role that promoters play in expression levels of genes of interest.
Figure 2: Library generation of genome-wide N’ yeast libraries using SWAT technology. a) Composition of the N’ SWAT cassette, containing the NOP1 promoter and GFP. This underwent swapping to the native promoter (and MTS or SP), and also to include the TEF2 promoter and mCherry. b) The SWAT library was also used to produce four further libraries as shown. Figure adapted from Yofe and Weil, et al. (2018)4
Another area of research that benefited from these new libraries, was the investigation of protein localisation. Using two libraries with different fluorophores enabled co-localisation studies to verify the correct localisation within the cell. Using GFP enabled the verification of 3,289 proteins with already assigned localisations, but also an additional 796 proteins could be assigned localisations from this study. This does not include the 544 proteins that were previously assigned in an earlier study, giving a total of 1,340 protein localisation assignments from these libraries.
Similarly, identifying protein locations may give rise to further information on the proteome. These libraries have been used to investigate the mitochondrial proteome, through isolating which proteins are present within mitochondria. Using an N’ SWAT library with the cassette containing a synthetic mitochondrial targeting sequence (MTS), and GFP, located 15 nt downstream of the original MTS CLEAVAGE site. This enabled the GFP to remain fused after MTS cleavage, and allowed visualisation of protein localisation. Through this, 10 new proteins were identified as mitochondrial; the genes encoding these proteins were mostly of an unknown function or name.
A further use for SWAT libraries is in investigating protein-protein interactions. Through a protein-fragment complementation assay (PCA), interactions can be visualised and qualitatively identified. Two PCAs were used, the first used split dihydrofolate reductase (DHFR); an enzyme that confers resistance to methotrexate. Splitting DHFR into two parts requires the two proteins to interact to enable DHFR production, and this can be seen by resistance to methotrexate, as shown in Figure 3. From the library interactions, there were 230 positive results, of which 165 had a paired set-up and 120 of these were reproducible. 109 of these interactions were unique and only 55.9% of these had been previously reported.
Figure 3: Protein-fragment complementation assays used to visualise protein-protein interactions. Resistance to methotrexate is conferred through DHFR activity, utilising split DHFR into F[1,2] and F allows protein-protein interactions to be investigated. If the two proteins do not interact (left), then the split fragments do not interact, inhibiting methotrexate resistance. If however, the two proteins do interact (right), then the split fragments will also interact and confer resistance to methotrexate.
Another example of a PCA used the fluorophore Venus, split into two parts, with libraries being tagged at the N’ terminus with either half of the complementary fragments. When crossed, this caused reconstitution of Venus and therefore visualisation of fluorescence. Interestingly, due to the affinity of the Venus fragments, they can be used to study membrane protein topology. Only membrane proteins with a Venus tag facing the cytosol will form a complete Venus molecule with the cytoplasmic half of the Venus tag, as shown in Figure 4. This meant that any fluorescence indicated by the split pair was located in the cytosol, defining the topology of the membrane protein.
Figure 4: Split Venus analysis to determine topology of N’ proteins. Schematic showing the mating of the N’ TEF2pr-VIC library, with a cytosolic VN strain. This caused a fluorescent signal only if the N’ of a membrane protein faced the cytosol. Figure adapted from Yofe and Weil, et al. (2018)4
In only a short period of time since the release of the first SWAT protocol, these libraries have shown their benefit across a wide range of current biological research. The uptake and use of tagged libraries can only enhance current research through more economical manipulation of large libraries. The SWAT libraries also encode potential barcodes since the linker region between each tag and its fusion protein is unique. utilizing this would allow for the study of several strains in a pooled fashion. This again increases the number of applications that this methodology is beneficial for.
The ease of swapping in a fraction of the time, and cost, enables any yeast laboratory to undertake large library analysis to systematically explore any protein.
1. Khmelinskii, A., Meurer, M., Duishoev, N., Delhomme, N. & Knop, M. (2011) Seamless Gene Tagging by endonuclease-Driven Homologous Recombination. PLoS ONE 6: e23794.
2. Yofe, I., Weill, U., Meurer, M., Chuartzman, S., Zalckvar. E., Goldman, O., Ben-Dor, S., Schütze, C., Wiedermann, N., Knop, M., Khmelinskii, A. and Schuldiner, M. (2016) One Library to make them all: Streamlining yeast library creation by a SWAp-Tag (SWAT) strategy. Nature Methods 13: 371-378.
3. Engel, S. and Cherry, J. The new modern ear of yeast genomics: community sequencing and the resulting annotation of multiple Saccharomyces cerevisiae strains at the Saccharomyces Genome Database. Database (Oxf.) 2013, bat012 (2013).
4. Weil, U., Yofe, I., Sass, E., Stynen, B., Davidi, D., Natarajan, J., Ben-Menachem, R., Avihou, Z., Goldman, O., Harpaz, N., Chuartzman, S., Kniazev, K., Knoblach, B., Laborenz, J., Boos, F., Kowarzyk, J., Ben-Dor, S., Zalckvar, E., Herrmann, J., Rachubinski, R., Pines, O., Rapaport, D., Michnick, S., Levy, E. and Schuldiner, M. (2018) Genome-wide SWAp-Tag yeast libraries for proteome exploration. Nature Methods 15: 617-622.
5. Meurer, M., Duan, Y., Sass, E., Kats, I., Herbst, K., Buchmuller, BC., Dederer, V., Huber, F., Kirrmaier, D., Štefl, M., Van Laer, K., Dick, TP., Lemberg, MK., Khmelinskii, A., Levy, ED., Knop, M. (2018) Genome-wide C-SWAT library for high-throughput yeast genome tagging. Nature Methods 15: 598-600.
“The fun never ends! Each Lab’s imagination is the only limitation.”
Prof. Maya Schuldiner – 2017