High-Throughput Yeast Library Construction

10th August 2017

Amber Leckenby1
1 Singer Instruments, Roadwater, Somerset, TA23 0RE, UK

INTRODUCTION

Systematic libraries have proven to be invaluable to genome-wide studies within yeast – examples include the yeast knockout collection and the yeast GFP library (find more libraries here). Each library has enabled novel insight into all aspects of yeast biology yet, their value is often overshadowed by the enormous effort required to make them. The large cost and lengthy laborious workflow of endless transformations, clone picking and validation steps often deter scientists from tackling other emerging biological questions.

Anton Khmelinskii and Matthias Meurer in Michael Knop’s lab at the University of Heidelberg developed a seamless gene tagging method[1] to help alleviate the problems faced during library construction. Together with Ido Yofe and Uri Weill in Maya Schuldiner’s lab at the Weizmann Institute of Science, Israel, they have further developed this method into the SWAp-Tag method. By utilising the ROTOR, this method allows rapid library construction in just three weeks [2].

Maya Schuldiner - Weizmann Institute of Science
Prof. Maya Schuldiner Weizmann Institute of Science, Israel

SWAp-Tag (SWAT) TECHNOLOGY

The SWAp-Tag (SWAT) method relies on the generation of an initial acceptor library. This acceptor library acts as a template that can be swapped into other libraries of choice in a “plug and play” manner (Figure. 1).

The library construction is unique in the fact that it requires a one-off traditional construction of an acceptor strain library with an acceptor module inserted at a known genomic location. This acceptor module can be replaced by a new tag, promoter or other desired genomic sequence by mating of the acceptor library with a donor strain expressing the desired module. The result is an unlimited number of new libraries created easily, accurately and cost-effectively.

 

Figure 1. The SWAT strategy enables rapid and straight-forward generation of systematic libraries. (a) Integration of the SWAT acceptor module to tag proteins at the N’ terminus. (b) Utilisation of the ROTOR to mate the acceptor strains with donor strain to create library of choice. Further utilisation of the ROTOR to select for haploid spores and induce I-SceI expression. (c) Examples of potential donor plasmids (top) that could be used to create gene tagged libraries (bottom).

 

SWAT TECHNOLOGY IS FAST, FLEXIBLE AND FREELY AVAILABLE

The Schuldiner and Knop labs have created an innovative and extremely rapid method for library construction by generating a library that has the ability to incorporate different modules easily, instead of creating a new library from scratch for each module. After acquisition or construction of an acceptor library, a new library can be generated in just three weeks and can be immediately used in a wide range of genome wide studies. The Schuldiner lab have already created an original acceptor N’-SWAT library which is N-terminally tagged with constitutively expressed GFP. In addition, two more libraries have been made from the original acceptor library using the SWAT method: an N’ mCherry-tag library and an N’ seamless GFP library. The published SWAT library is now freely available in GFP-tag, mCherry-tag or seamless GFP flavour from Prof. Maya Schuldiner.

The bottlenecks in this method are the mating and selection steps. The Schuldiner and Knop labs were able to use the Singer Instruments ROTOR to alleviate these bottlenecks. As such, the ROTOR was used for all handling of strains, mating and sporulating procedures, tag-swap selections/counter-selection and in library screens for the selection of successful module integration.

 

“Creation of new libraries would not have been possible without our Singer ROTOR robot”.
Prof. Maya Schuldiner – 2017

 

This swift process means that scientists can afford to be flexible in the libraries that they create and visualise never before seen proteins. Tag-mediated localisation problems have been solved by the SWAp-TAG method as it enables ORFs to be tagged at either the 5’ or 3’ end to minimise any mis-localisation or protein-destabilisation effects.

 

WHAT’S NEXT FOR THE SWAT STRATEGY: APPLICATIONS AND IMPLICATIONS?

Theoretically, any tag can be used to construct a new library from this N’-SWAT acceptor library. This tag could be different coloured fluorophores for co-localization studies or complementation tags for measuring protein-protein interactions. Once a C’-SWAT acceptor library has been generated, any section of the 5’- or 3’-end of a gene can be modified whether it be a promoter, UTR or other non-coding DNA to quantify transcription and translation effects. Half-lives can be studied using timer fluorophores or a pull down tag can be attached to isolate proteins.

 

“The sky is this limit and now only each labs’ imagination is the problem”.
Prof. Maya Schuldiner – 2017

 

The SWAT method for library generation frees up valuable time, allowing researchers to design impactful experiments. The Schuldiner lab are already using the N’ SWAT library in co-localization screens, overexpression screens and for looking at interactomes in vivo. As Professor Schuldiner has said herself, “The fun never ends!”.
 
Discover how the ROTOR can help you too!
 
 
ROTOR HDA High-throughput Screening Robot
 
 

REFERENCES

  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. Huh, WK., Falvo, JV., Gerke, LC., Carroll, AS., Howson, RW., Weissman, JS. and O’Shea, EK. (2003) Global analysis of protein localization in budding yeast. Nature 425: 686-91

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