Category Archives: ROTOR

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.


Surprise for 100th ROTOR customer

When Dr. Matthias Peter saw his glistening new ROTOR High Density Array robot installed in his laboratory at ETH Zurich, he did not expect to see his head on the body of a three-armed budding cerevisiae cell staring back at him. But…that’s exactly what he got!

With the help from his lab tech Ingrid Stoffel and her colleagues, we surprised our 100th worldwide ROTOR installation, Dr. Peter, with a custom graphic of the budding yeast version of Dr. Peter thinking about a triangle?!

“The triangle is a running joke in our lab for so many years”, Ingrid explains, “Matthias always has the same starting slide showing a triangle for all his presentations. In our group we have three subgroups, so the triangle is like a symbol representing all of us.”

High-throughput Screening Peter Group – Institute of Biochemistry ETH Zurich, custom ROTOR HD

 

“He had the big smile on his face.”, Ingrid recalled when Dr. Peter first saw the robot. “There was a queue of people trying to have a look!”

 

“I was surprised and I love it”, said Caroline, another lab member, as she cracked into laughter again, “it shows that we can be scientists, but still have a sense of humour”

 

High-throughput Screening Peter Group – Institute of Biochemistry ETH Zurich ROTOR High-throughput screening robot

 

The Peter lab are interested in how cell growth and division are regulated in space and time. The robot enables them to systematically identify all components in the protein degradation pathway by using a methodology called SGA (Synthetic Genetic Array).

 

“It’s at that fantastic starting point to start with the screen. For example, we work on translation at the moment, so I could make a smaller collection of all the ribosomal proteins and test them under various conditions. I mean it just broadens my options massively”, said Caroline, who is spearheading the SGA screens at ETH.

 

“The user interface is really intuitive as soon as we switched it on, we could easily figure out how to use the robot because it has step-by-step instructions.”

 

Ingrid, who is also doing the screen with Caroline added, “It’s almost impossible to crash it!”

 

High-throughput Screening ROTOR High-throughput screening colony re-arraying robot at ETH Zurich

 

We believe in our responsibility to science, and are proud to have accelerated research in 100 laboratories, in 26 countries, across 5 continents. We know each robot can easily manipulate 100,000 colonies in an hour. Assuming the average run time on the robot is merely 3 hours per day, these 100 robots alone will collectively help us screen through more than 10 billion colonies in the next year and continue to accelerate the speed with which we cure cancer, discover antibiotics, and engineer ethanol-producing super bacteria!

 

We have no doubt that this robot will bring more laughter and success to not just Dr Peter’s lab, but also other microbiology labs at ETH, just like the 99 other robots that came before it did.

High-throughput Screening ROTOR High-throughput screening colony re-arraying robot at ETH Zurich

 

To all 100 robots out there that help accelerate and unite microbial screening labs around the world, and not to forget our Service & Support Manager Ian who installed the majority of them! Proscht!

 

See how high-throughput microbial pinning can help you!

 

 

 

 

Underlying causes of Parkinson’s disease

New publication uses Singer Instruments’ ROTOR to better understand underlying causes of Parkinson’s disease by risk genes VPS35 and EIF4G1.

Who/Where: Dhungel et al., 2015, Neuron, Volume 85, Issue 1, 7 January 2015, Pages 76-87

Preview of paper: www.cell.com/neuron/abstract/S0896-6273(14)01154-4

 

By: Aaron D. Gitler

The Gitler Laboratory, Stanford University, CA, USA

gitlerlab.googlepages.com

 

Parkinson’s disease is a relatively common neurodegenerative movement disorder caused by a loss of dopamine-producing neurons from the substantia nigra region of the brain. The disease is mostly sporadic but about 10% of cases exhibit a familial pattern of inheritance and these rarer cases have led to the identification of mutations in genes that can cause Parkinson’s disease. Studying the functions of these familial Parkinson’s disease genes have led to important insights into the cellular pathways that are affected in even the more common sporadic forms of the disease. A better understanding of the pathways that contribute to Parkinson’s disease will hopefully lead to more effective therapeutic approaches.

 

In our laboratory we have been using a very simple experimental model system to study human diseases associated with protein misfolding and neurodegeneration. We are using the budding yeast, Saccharomyces cerevisiae. Even though human diseases like Parkinson’s disease are very complicated, we hypothesize that some of the defects that underlie the disease are associated with alterations in basic cellular pathways, and nearly all of the key cellular pathways are well-conserved from yeast to human. By using the power of yeast genetics, our laboratory aims to uncover mechanisms associated with terrible disorders such as Parkinson’s disease.

High-throughput Screening Better understand underlying causes of Parkinson’s disease by risk genes VPS35 and EIF4G1.

 

Two of the newer genes implicated in Parkinson’s disease are EIF4G1 and VPS35. EIF4G1 encodes a translation initiation factor and VPS35 is component of the retromer complex. EIF4G1 and VPS35 both have related genes in yeast (human EIF4G1 = yeast TIF4631 and TIF632 and human VPS35 = yeast VPS35). Since both of the yeast homologs of EIF4G1 and VPS35 are non-essential genes, strains of yeast with deletions of either were alive and growing well. We wanted to define the cellular functions of these genes so we decided to perform a screen to identify genes that we could delete in combination with either Tif4631 or Vps35 that would cause a growth defect. This type of a screen is called a synthetic lethal screen and is a powerful way to identify genes that function in similar cellular processes. It gives us great insight into how the genes interact with the rest of the genome, thereby giving us clues as to the pathways that are perhaps involved in Parkinson’s disease.

High-throughput Screening Better understand underlying causes of Parkinson’s disease by risk genes VPS35 and EIF4G1.

 

The yeast genome contains about 6,000 genes and about 4,850 of these are non-essential. Manipulating those many strains manually is a tremendous task that is nearly impossible and time consuming. Luckily we were able to utilize the Singer Instruments ROTOR, a High Density Array robot to conduct the monumental screens. This automated robotics method was used to combine each of the 4,850 non-essential haploid yeast deletions with either the vps35 deletion or the tif4631 deletion by mating. This generated diploid cells, which we then sporulated and finally selectively germinated haploid progeny that contained both deletions. All of these tedious tasks were taken care of by our Singer Instruments ROTOR and we did three replicates of the screens. We then used image analysis software to compare the size of colonies in order to identify double mutants that grew worse when compared to either of the single mutants. This gave us great insight into how VPS35 and EIF4G1 functioned. We were then able to take the yeast data and confirm our findings in other model organisms such as the nematode worms and mice. By using the ROTOR, we were able to conduct large genome-wide screens in less than two weeks and produce reliable, statistically testable, and repeatable data.