Name: High-throughput Yeast Plasmid Overexpression Screen (Video) | JoVE Journal
Uploaded: 2011-07-27T13:00:00.000+00:00
Duration: 8 min 57 s
Description: Learn a high-throughput yeast transformation protocol using a liquid handling robot to screen plasmid libraries in Saccharomyces cerevisiae. This method identifies genetic modifiers of neurodegenerative disease protein toxicity.

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5 minutes

This work was supported by a grant from the Packard Center for ALS Research at Johns Hopkins (A.D.G.), an NIH Director&#x2019;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.

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.
<ol>
<li>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 CEN and 2μ yeast plasmids.</li>
<li>Inoculate 200mL of yeast peptone dextrose (YPD) media (10g/L yeast extract, 20g/L peptone, 20g/L glucose) with query strain in a 500mL baffled Erlenmeyer flask. Incubate overnight at 30°C with shaking (200 rpm). If required, selective media can also be used for these starter cultures.</li>
<li>The following morning, dilute the query strain to OD600=0.1 in 2L of YPD. Shake for 4-6 hours at 30°C (200 rpm) until the culture reaches OD600=0.8-1.0. For optimal growth, grow up two 1L cultures in separate 2.8 L baffled flasks. If required, selective media can also be used instead of YPD.</li>
</ol>
2. Yeast transformation
<ol>
<li>Harvest cells by centrifugation. Fill four disposable 225mL conical tubes with yeast culture and spin at 3000rpm (˜1800 x g) for 5 minutes in tabletop centrifuge (Eppendorf 5810R). Pour off supernatant and repeat until all cells are harvested.</li>
<li>Wash cells in 200mL of autoclaved distilled H2O. Resuspend cell pellet by shaking or vortexing. Combine cells in one 225mL conical tube. Spin at 3000rpm for 5 minutes. Remove supernatant.</li>
<li>Prepare 0.1MLiOAc/1XTE solution (0.1M LiOAc, 10mM Tris, 1mM EDTA, pH 8 in autoclaved distilled water). Wash cells in 100mL 0.1MLiOAc/1XTE. Spin at 3000rpm for 5 minutes. Remove supernatant.</li>
<li>Resuspend cell pellet in 70mL 0.1M LiOAc/1xTE. Shake and/or vortex to ensure pellet is completely resuspended.</li>
<li>Incubate with shaking at 30°C for 30 minutes (200 rpm).</li>
<li>Add β-mercaptoethanol to 0.1M. Incubate with shaking at 30°C for 30 minutes (200 rpm). Note: This step can be omitted if using yeast strains that are sensitive to β-mercaptoethanol. We have found that this step is not essential for successful transformation, however it does improve transformation efficiency.</li>
<li>Boil 2mL of sonicated salmon sperm DNA (10mg/mL) for 15 minutes, and then chill on ice.</li>
<li>Add 2mL of boiled salmon sperm DNA to cell mixture. Caution: A precipitate may form upon addition of the salmon sperm DNA to the cell mixture. Using a pipetman and 200 μl tip, carefully remove any precipitate that forms because this may interfere with downstream pipetting.</li>
<li>Aliquot 50μL of cell mixture to each well of the 96-well plate using the BioRobot Rapidplate (can also use multi-channel pipette). Do not mix.</li>
<li>Incubate at room temperature for 30 minutes without shaking.</li>
<li>Prepare 200mL of 40%PEG-3350/10%DMSO/0.1M LiOAc (160mL of 50% PEG-3350, 20mL DMSO, 20mL 1M LiOAc). Prepare solution immediately before use.</li>
<li>Add 125μL of PEG/LioAc/DMSO solution to each well. Mix by aspirating and dispensing cell suspension eight times with RapidPlate.</li>
<li>Incubate at room temperature for 30 minutes without shaking.</li>
<li>Heat shock the cells at 42°C for 15 minutes in a dry incubator. Do not stack plates. Note: We have found that this heat shock step is not essential for successful transformation. For certain yeast strains (for example, temperature sensitive mutants), heat shock is deleterious. We have previously reduced the heat shock time to one minute and have been able to successfully transform a yeast strain harboring a ypt1 temperature sensitive mutation using this protocol.</li>
<li>Spin plates at 2500rpm (˜1100 x g) for 5 minutes, using centrifuge adapters to accommodate 96-well plates. Remove PEG solution by inverting plates over waste bucket. Blot inverted plate on paper towel to remove residual liquid (cells will remain on bottom of wells).</li>
<li>Rinse cells by adding 200μL of minimal media (e.g. SD/-Ura) to each well with RapidPlate.</li>
<li>Spin plates at 2500rpm for 5 minutes and remove supernatant by inverting over waste bucket and blotting on paper towels.</li>
<li>Add 200μL of minimal media (e.g. SD/-Ura) to each well with RapidPlate.</li>
<li>Incubate at 30°C for 2 days without shaking. After 2 days, small colonies of cells should begin to form at the bottom of each successfully transformed well.</li>
</ol>
3. Spotting assay
<ol>
<li>Dispense 200μL of raffinose based minimal media (e.g. SRaf/-Ura) into each well of a new flat-bottom 96-well plate.</li>
<li>Mix each well of transformation plate by aspirating and dispensing cell suspension eight times with Rapidplate.</li>
<li>Inoculate SRaf plates with 5μL of cell mixture from transformation plate.</li>
<li>Incubate at 30°C for one day. Small colonies should be present at the bottom of each well after one day.</li>
<li>Spot colonies on SD/-Ura and SGal/-Ura plates in hood. Sterilize 96-bolt replicator (frogger) by flaming. Mix colonies with frogger before spotting on selective media plates. After spotting, allow plates to dry in hood for 5-10 minutes.</li>
<li>Incubate at 30°C for 2-3 days.</li>
</ol>

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:
<img src="/files/ftp_upload/2836/2836fig1.jpg" alt="Microplate assay comparing TDP-43 expression in glucose and galactose, highlighting gene regulation." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
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.

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<li>Neumann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitinated+TDP-43+in+frontotemporal+lobar+degeneration+and+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.</a> Science. 314, 130-133 (2006).</li>
<li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+yeast+TDP-43+proteinopathy+model%3A+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proc Natl Acad Sci U S A. 105, 6439-6444 (2008).</li>
<li>Elden, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466, 1069-1075 (2010).</li>
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No conflicts of interest declared.

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...

<table><tbody><tr><td>BioRobot RapidPlate</td><td>Qiagen</td><td>9000490</td><td></td></tr><tr><td>96 bolt replicator (frogger)</td><td>V&amp;amp;P Scientific</td><td>VP404</td><td></td></tr><tr><td>FLEXGene ORF Library</td><td>Institute of Proteomics, Harvard Medical School</td><td></td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>500mL baffled flask</td><td>Bellco Glass</td><td>2543-00500</td><td></td></tr><tr><td>2.8L triple-baffled Fernbach flask</td><td>Bellco Glass</td><td>2551-02800</td><td></td></tr><tr><td>100&amp;mu;L Rapidplate pipette tips</td><td>Axygen Scientific</td><td>ZT-100-R-S</td><td></td></tr><tr><td>200&amp;mu;L Rapidplate pipette tips</td><td>Axygen Scientific</td><td>ZT-200-R-S</td><td></td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

High-throughput Yeast Plasmid Overexpression Screen

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.

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, ...

The overall goal of this procedure is to transform yeast cells with an array library of plasma DNAs. This is accomplished by first growing up an appropriate amount of yeast cells. The yeast cells are then treated with lithium acetate to make them competent for DNA transformation.Next, transform the competent e cells by mixing them with DNA. The final step of the procedure is to select and analyze transformants. Ultimately, results can be obtained that show the effects of the plasma DNA on yeast phenotypes through yeast spotting assays and or microscopy.The main advantage of this technique over existing protocols, such as standard yeast transformations, is that it allows for the transformation of a large number of different Plasmids into yeast cells in an automated fashion. This high throughput yeast Transformation protocol is designed for 10 96 well plates, but can be scaled up or down accordingly. For larger scale experiments, the key to success is to keep all plates and reagents organized.Once the experiment is underway to avoid confusion, A biobot rapid plate liquid handler will be used in this protocol To begin aliquot, five to 10 microliters of plasma DNA from an array yeast plasmid library into each well of the round. Bottom 96 well plates. Place the 96 well plates uncovered inside a clean bench fume hood overnight to dry.Next inoculate 200 milliliters of yeast peptide dextrose or YPD media with a query strain in a 500 milliliter baffled Lin Meyer flask incubate overnight at 30 degrees Celsius with shaking on the following morning. Dilute the query strain to an OD 600 of 0.1 in two liters of YPD media for optimal growth. Grow up to one liter cultures in separate 2.8 liter baffled flasks.Shake the flasks for four to six hours at 30 degrees Celsius until the cultures reach an OD 600 of 0.8 to 1.0. When the yeast Culture has reached an OD 600 of 0.8 to 1.0, harvest the cells by centrifugation. Fill four disposable 225 milliliter conical tubes with culture, and spin a 3000 RPM for five minutes in a tabletop centrifuge.Pour off the S supernatant from each tube. Add more culture to the same tubes and centrifuge again, repeating as necessary until all cells are harvested. Wash each cell pellet in 50 milliliters of autoclave distilled water and Resus spend by vortexing.Combine cells in one two hundred and twenty five milliliter conical tube and spin at 3000 RPM for five minutes. After removing the S supernatant, wash the cells in a hundred milliliters of lithium acetate solution. Spin again at 3000 RPM for five minutes.Remove the SNA resuspend the cell pellet in 70 milliliters of lithium acetate solution. Vortex at high speed for about 30 seconds until the pellet is completely resuspended. Incubate with shaking at 30 degrees Celsius for 30 minutes.Next, add beta me capto ethanol to 0.1 molar incubate with shaking at 30 degrees Celsius for another 30 minutes. After the 30 minute incubation, add two milliliters of boiled and chilled salmon sperm DNA to the cell mixture. Pour the cell mixture into an autoclavable single well plate, and then remove any precipitate that forms because this may interfere with downstream pipetting.Using the biobot rapid plate aliquot 50 microliters of cell mixture to each well of the four previously prepared. 96 well plates containing plasma DNA do not mix. Incubate the plates at room temperature for 30 minutes without shaking while the cells are incubating.Prepare 200 milliliters of PEG DMSO lithium acetate solution. Combine 160 milliliters of 50%PEG 33, 50 20 milliliters of DMSO and 20 milliliters of one molar lithium acetate. It is important that this solution is prepared just before use.Add 125 microliters of PEG lithium acetate DMSO solution to each. Well mix by aspirating and dispensing the cell suspension eight times. Incubate at room temperature for 30 minutes without shaking.Next, heat shock the cells at 42 degrees Celsius for 15 minutes In a dry incubator, do not stack the plates after the heat shock, spin the plates using centrifuge adapters to accommodate the 96 well plates. After centrifugation, remove the PEG solution by inverting the plates over a waste bucket. Then blot the inverted plates on paper towels.To remove residual liquid, the cells will remain on the bottom of the wells. Rinse the cells by adding 200 microliters of minimal media to each. Well spin the plates again.Remove the supernatant by inverting over a waste bucket and blotting on paper towels. Add 200 microliters of minimal media to each well with a rapid plate incubate at 30 degrees Celsius for two days without shaking. After two days, small colonies of cells should begin to form at the bottom of each successfully Transformed.Well to prepare for the Spatting assay, first dispense 200 microliters of OSE based minimal media into each well of a new flat. Bottom 96 well plate.Next. Mix each well of the transformation plate by aspirating and dispensing the cell suspension eight times with a rapid plate.Inoculate each well of the raffinose plate with five microliters of cell mixture from the transformation plate, incubate at 30 degrees Celsius for one day. On the following day. Small colonies should be present at the bottom of each.Well perform the spotting assay in the hood, sterilize a 96 bolt, replicator or frogger by flaming. Mix the colonies with a frogger and then spot colonies on selective media plates. After spotting, allow the plates to dry in the hood for five to 10 minutes.Incubate the plates at 30 degrees Celsius for Two to three days. Shown Here are representative results of a yeast plasmid over expression screen to identify suppressors and enhancers of TDP 43.Toxicity. TDP 43 is a human protein that has been implicated in the pathogenesis of a myotrophic lateral sclerosis or Lou Gehrig's disease expressing TDP 43 in yeast cells results in aggregation and cytotoxicity.These plates display colonies with an integrated galactose inducible, T DP 43 plasmid that have also been transformed with plasmids from the flex gene ORF expression library. The colonies here appear red in color because the strain happens to have a mutation that results in accumulation of a red pigment. The plate on the left contains glucose, which represses expression of T DP 43 or the flex gene plasmids.The plate on the right contains galactose, which induces the expression of TDP 43 and the O Fs in the flex gene plasmids. The green arrowhead indicates a colony transformed with a plasmid that suppresses the toxicity of TDP 43 as indicated by faster growth and more dense colonies. The red arrowhead indicates a colony transformed with a plasmid that enhances the toxicity of TDP 43 as indicated by slower growth and less dense Colonies.After watching this video, you should have a good understanding of how to transform an avoid library of plasma DNAs de cells.

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Learn a high-throughput yeast transformation protocol using a liquid handling robot to screen plasmid libraries in Saccharomyces cerevisiae. This method identifies genetic modifiers of neurodegenerative disease protein toxicity.

High-throughput Yeast Plasmid Overexpression Screen

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