High-throughput Yeast Plasmid Overexpression Screen

Summary

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.

Abstract

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.

Protocol

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.

1. Aliquot 5-10μL of plasmid DNA (100 ng/μl) from the Yeast FLEXGene ORF library into each well of a round-bottom 96-well plate with Biorobot RapidPlate liquid handler. Place uncovered 96-well plates in clean bench fume hood overnight to dry. We have found similar transformation efficiencies with CE....

Discussion

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 assoc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 Glass	2543-00500
2.8L triple-baffled Fernbach flask	Bellco Glass	2551-02800
100μL Rapidplate pipette tips	Axygen Scientific	ZT-100-R-S
200μL Rapidplate pipette tips	Axygen Scientific	ZT-200-R-S