Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.
1. Preparations for yeast transformation
This protocol is designed for ten 96-well plates but can be scaled up or down accordingly. We have found that this protocol does not work well for more than twenty 96-well plates per round of transformation. The entire transformation procedure (from step I.3) will take approximately eight hours.
2. Yeast transformation
3. Spotting assay
Photograph plates with digital camera, and visually compare growth of colonies on SGal/-Ura plates to find colonies in which query strain toxicity is enhanced (slower growth/less dense colonies) or suppressed (faster growth/more dense colonies).
4. Representative results:
Figure 1. Yeast plasmid overexpression screen to identify suppressors and enhancers of TDP-43 toxicity. TDP-43 is a human protein that has been implicated in the pathogenesis of amyotrophic lateral sclerosis (Lou Gehrig’s disease). Cytoplasmic aggregates of TDP-43 accumulate in the brain and spinal cord neurons of ALS patients 17. Expressing TDP-43 in yeast cells results in aggregation and cytotoxicity 18. We have used this model system to define mechanisms of TDP-43 toxicity 19,20. Shown are repesentative examples of plates from our yeast TDP-43 toxicity modifier screen. These plates display colonies with an integrated galactose-inducible TDP-43 plasmid and also transformed with plasmids from the FLEXGene ORF expression library. The plate on the left contains glucose, which represses expression of TDP-43 or the FLEXGene plasmids. The plate on the right contains galactose, which induces the expression of TDP-43 and the ORFs in the FLEXGene plasmids. The green arrowhead indicates a colony transformed with a plasmid that suppresses the toxicity of TDP-43. The red arrowhead indicates a colony transformed with a plasmid that enhances the toxicity of TDP-43.
Here we present a protocol to perform a high-throughput plasmid overexpression screen in yeast. This approach allows for the rapid and unbiased screening for genetic modifiers of many different cellular phenotypes. Using this approach, a researcher can screen a significant portion of the yeast genome in a matter of weeks. This unbiased approach also allows for the identification of modifiers, which may not have been predicted based on previous findings. We have used this approach to identify modifiers of toxicity associated with the aggregation of human neurodegenerative disease proteins 19,21-23. However, owing to the adaptability of this protocol to study other cellular processes, this protocol will be useful to researchers addressing a wide range of important biological questions. For example, we have also found this screening approach useful for identifying genes and pathways involved in adaptation to environmental stressors, including heavy metals and oxidative stress. Here we have focused on one plasmid library, the Yeast FLEXGene library 9. However, there are several other yeast plasmid libraries available, which could also be used for these screens 6,8.
The authors have nothing to disclose.
This work was supported by a grant from the Packard Center for ALS Research at Johns Hopkins (A.D.G.), an NIH Director’s New Innovator Award 1DP2OD004417-01 (A.D.G), NIH R01 NS065317 (A.D.G.), the Rita Allen Foundation Scholar Award. A.D.G. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts.
Name of reagent | Company | Catalog number |
---|---|---|
BioRobot RapidPlate | Qiagen | 9000490 |
96 bolt replicator (frogger) | V&P Scientific | VP404 |
FLEXGene ORF Library | Institute of Proteomics, Harvard Medical School | |
Tabletop centrifuge | Eppendorf | 5810R |
500mL baffled flask | Bellco | 2543-00500 |
2.8L triple-baffled Fernbach flask | Bellco | 2551-02800 |
100μL Rapidplate pipette tips | Axygen | ZT-100-R-S |
200μL Rapidplate pipette tips | Axygen | ZT-200-R-S |