Quick and easy bioengineering procedures: Yeast (S. cerevisiae) transformation
This is the third post in a series of 4 in which I want to share a quick and easy to use procedure for engineering a yeast strain. The idea is to go from the cloning stage, right after doing a PCR (or order) to get the DNA you want to clone, up to the process of transforming the yeast and selecting the positive clones. Keep in mind that the goal here is to make it quick and easy, so I'm targeting those who are not experimental biologists and want minimal lab work, or those who have a super wonderful, really cutting edge protocol, but maybe go overwhelmed in terms of time and resources and want to simplify the cloning and transformation stages of the project.
In this post I share a simple and efficient procedure to incorporate exogenous DNA into Saccharomyces cerevisiae cells, whether the goal is to integrate DNA into the chromosome or to introduce an episomal plasmid. In the context of this series of posts, this procedure would come after obtaining the DNA intended to be transformed into yeast from a miniprep of the E. coli cells generated in the second post. The procedure presented here is known as lithium acetate (LiAc)/single-stranded carrier DNA/polyethylene glycol (PEG) transformation. There are other ways to transform yeast, and in particular Saccharomyces cerevisiae, so depending on the type of yeast and the complexity of the transformed DNA, the most appropriate approach (spheroplast, electroporation, biolistics, ...) will have to be chosen. In my experience, I have used this method on strains derived from the widely used laboratory strain S288C (i.e. BY4742, 41 ...), to integrate linear DNA chunks up to 5Kbp, and plasmids up to 10Kbp. There are probably countless versions of the protocol presented below, this particular one was derived from Gietz, 2014.
The principle of this method is not fully demonstrated, but most models are based on the use of PEG together with lithium cations to attach the DNA to the cell wall and penetrate it, respectively, all assisted by heat shock. In addition, the presence of single-stranded carrier DNA, usually from salmon sperm (don't ask me why salmon, but better to leave it like this), turns out to be essential in my experience, and is most likely used to prevent foreign DNA from being degraded by nucleases inside the cell.
Some detail to keep in mind for the success of this method, and indeed whenever working with yeast, is that homologies of foreign DNA to the yeast genome will potentially lead to unwanted recombination, especially if the introduced DNA is linear. Everything can become a mesh if this is not taken into account. Therefore, try to avoid DNA sequence homologies unless you want to use them to integrate DNA into the chromosome. However, if that is the case, it is most efficient to transform the piece of DNA targeted for integration into the linear state, after restriction with appropriate enzymes in a separate reaction.
Once your DNA is ready to be transformed, whether it is a circular plasmid or a linear piece from a PCR or restriction reaction, the method is based on washing the cells from mid-exponential phase with water, then with LiAc, and mixing with PEG, single-stranded carrier DNA, and the desired DNA to transform. The mixture is then incubated at 30°C for 30 minutes, followed by another incubation for one to two hours at 42°C. After this, transformation should have occurred, and what follows is recovery in rich YPD medium at 30°C for two hours, and selection on plates. The procedure can take a day (excluding selection), and a couple of hours of dedicated work, but allows several reactions to be carried out in parallel, and without the need for competent cells.
PROTOCOL FOR YEAST TRANSFORMATION:
The night before grow the yeast strains to transform in 5mL of YPD at 30 °C. From the overnight culture, dilute 100 times into 50mL of YPD fresh media and let it grow for 4 hours at 30 °C, shaking. (The OD600 should be between 0.3-0.4). Then follow this procedure:
- Harvest the cells by pelleting at 3000 rpm in a centrifuge for 10 minutes at room temperature and discard the supernatant (seems a lot? They always survive)
- In parallel, place a thawed tube of carrier DNA reagent in a boiling water bath for 5 minutes and chill immediately in ice.
- Resuspend the cells in 10 mL of pure water
- Harvest the cells by pelleting at 3000rpm in a centrifuge for 10 minutes at room temperature and discard the supernatant
- Resuspend the cells in 1 mL of 100mM LiAc and transfer to a 1.5 mL tube
- Pellet the cells at 3000 rpm for 10 minute, remove the supernatant, and resuspend the pellet in 400 µL of 100 mM LiAc
- Aliquot 50 µL of cell suspension into 1.5 mL tubes (One per transformation and controls)
- Pellet the cells at 3000 rpm for 1 minute and remove the supernatant
- Add to the pellet, IN THE ORDER GIVEN, 240 µL PEG 3350 (50% w/v), 36 µL LiAc (1.0M), 50µL single-stranded carrier DNA (2.0 mg/mL) and 34 µL of sterile water plus any plasmid DNA (up to 1 µg if it is episomal plasmid, and 1-4µg if it is linear DNA for integration), and mix the pellet by vortex mixing briskly until resuspended, no clumps (usually I transform directly the restriction product if it is the case)
- Incubate for 30 minutes at 30 °C
- Incubate the tube in a water bath at 42 °C for at least one or two hours. Temperature-sensitive strains can be left on the bench overnight, then carried on to the next step
- Centrifuge the transformation tube at 3000rpm for 1 minute at room temperature and remove the transformation mix with a pipette
- Resuspend by pipetting gently in 1 mL of YPD and incubate two hours at 30°C shaking.
- Harvest the cells by centrifuging at 3000 rpm 10 minutes
- Resuspend the cells in 200 µL of sterile water by pipetting gently
- Plate the cells suspension on selectable plates (20 µL in one plate, 180 µL in the other)
Then, select the positives and check them by colony PCR for yeast, that is a bit subtler than for bacteria. Basically, the lysis is done with NaOH during 20 minutes at 90C, and the maximal recommended length for the amplicon is of around 1kb. But I will explain you this in the next post.
REFERENCES: Gietz, R. D., & Woods, R. A. (2006). Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Yeast Protocol, 107-120.
Hey Sebastián! Have you ever determined whether the extended heat shock (1 or 2h) affects overall efficiency? A while back I was trying to do this protocol in sort of a "bulk" setup, where I just increased all of the reagents to a certain factor so that I could essentially do like 50 transformations all in one tube (to increase the number of colonies), rather than aliquoting them out as you do in Step 7. However, when I would heat-shock the tube I think the heat wasn't getting distributed evenly in it (given it was like 40mL of volume), because the resulting efficiency was awful. Let me know your thoughts!