Category Archives: High Throughput Screening

High-Throughput Yeast Library Construction

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

Grow Your Own GFP Xmas Tree

Is it Christmas yet? The Singer lab has certainly been getting into the festive spirit. The flasks are full of sherry and the turkey’s in the incubator! All that’s left to sort out is the Christmas tree. But, if like us, you’re sick of untangling the tree lights and cleaning up the constant shower of pine needles, we have the ultimate solution: grow a GFP Christmas tree!
 


 


To create your own GFP Christmas tree you will need:

 

Ready? Let Christmas commence!

 
1. Load the Stinger file into the ROTOR.
 
2. Follow the on-screen instructions for loading your 384-density, GFP source plate and the target plate, then hit go!
 
3. Drink some sherry and be merry while the Stinger does its thing.
 
4. Use the PhenoBooth to watch the GFP tree glow and come to life.
 

Download our Stinger template file and get pinning!
We’d love to see how yours turn out. Share your fluorescent colonies on our Facebook / Twitter pages using the hashtag: #gfpxmas
 

Merry Christmas to all and to all a good science.


Agar Plate Pourer (Competition)

Currently the Serial Filler is unavailable. We have received a lot of fantastic feedback and improvement suggestions from existing Serial Filler customers. Rather than investing in the next large Serial Filler production run, Singer Instruments are in the process of considering an improved model.

…And after much (a couple of years) tribulation, we are proud to announce that the winner is Dan Smethurst from the Campbell Gourlay lab, in the Kent Fungal Group at the University of Kent!

Watch the moment we ‘surprised’ Dan Smethurst with his fabulous new Automatic Agar Plate Pourer – the Serial Filler:

Deep question: What’s my role in the World? What will be my scientific legacy? What is the answer that I am devoting my entire academic life and therefore soul into finding?

Maybe its curing cancer, explaining prion protein misfolding, or preventing death?! Maybe its understanding the role BCL2 plays in ear colour or some other hypothesis so niche that three years later you begin to doubt whether you can even buy a can of ravioli from the corner shop without sweating profusely and mumbling inanely about stop codons.

You’ve spent years producing network diagrams, learning how to spell Aduncuperistomatus or trying to understand why the hell you need to comb DNA. The bottom line is that you are bloody clever! And with this being true, you should be spending your high functioning brainpower doing bloody clever things! As such we reached one vehemently emphatic conclusion:

Pouring plates… what a f**king waste of time!

Solution: We have made a kick-ass plate pourer.

But with Slime Mold marginally edging the intelligence scale when pitted against our Product Development team, we realised that coming up with a name ourselves again (like we did with the spectacularly creative MSM 100, 200, 300 and then 400 microscopes) was probably not the greatest idea.

But wait! Fear thee not! We know scientists! And scientists are clever! Three months of blood, sweat and coffee later, and triumphantly our team devised an intrepid plan:

Microbiologist + Competition = Good name for plate pourer

Singer Instruments’ scientific advisory team stepped in and highlighted a chemical imbalance, and made one small alteration:

Microbiologist + Competition = Good name for automatic plate pourer + Free plate pourer for winner

Here are but a handful of the wonderful entries that we received:

Thanks very much to everybody who took part! You are all legends!