Guide to Making and Storing Competent Yeast Cells
Yeasts, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, are routinely used in biology research labs around the world. Yeasts are easy-to-culture, unicellular eukaryotes, and make excellent model organisms because of the similarity of their genes and proteins with those of their mammalian counterparts.
Yeast cells are used to study gene function, protein interactions, and cellular pathways. They are also excellent hosts for large-scale heterologous protein expression and small molecule production, and are a mainstay in synthesis labs, as well as in the baking, brewing, and pharmaceutical industries. Common experimental setups involving yeast include the preparation of yeast one/two/three-hybrid systems, generation of mutant screening libraries and homologous recombination, to name a few.
Why Do We Need Competent Yeast Cells?
The setups mentioned above involve the introduction of foreign DNA into yeast at some point during the experimental process. Introduction of gene fragments (linear ssDNA or plasmids) is achieved via transformation of yeast cells either through chemical methods (e.g., lithium acetate and polyethylene glycol (PEG)) or through electroporation.
Importantly, efficient yeast transformation requires starting out with high quality competent yeast cells. Competency refers to the ability of cells to take up free, extracellular genetic material (e.g., plasmid DNA). This article will tell you exactly how to achieve this and, hopefully, set you well on your way to yeast transformation success!
Preparing Competent Yeast Cells
The protocol to acquire competent cells is fairly straightforward, but many researchers run into problems with achieving good survival ratios and transformation efficiencies. Common pitfalls include high cell death due to harsh preparation conditions, or poor transformation efficiencies due to ineffective competent cell preparation.
Although yeast cells are as easy to grow and transform as E. coli, they require different treatment for competent cell preparation and transformation. Here, yeast cells are treated like mammalian cells with similar procedures and care. A typical competent cell preparation protocol for yeast is as follows:
- Culture the yeast strain(s) you want to transform overnight. Use nutrient rich medium to grow cells that do not harbor plasmids. For cells already containing plasmids, use appropriate selective media to maintain the plasmid.
- Harvest cells by centrifugation once they reach the right cell density (usually 1 to 2 x 107 cells/mL). You can either measure cell density using a spectrophotometer at OD600, or by counting cells under the microscope using a haemocytometer.
- Wash cells by resuspending them in sterile water and centrifuge to discard water with any traces of medium. Repeat this wash step twice more to wash cells thoroughly.
- After the final wash, suspend the cell pellet in a competent cell solution* and aliquot about 108 cells per tube (each transformation reaction will use these number of cells). Freeze tubes at -80°C (more details on storage below). This will give you a stash of competent yeast cells to use in future experiments.
- In all steps, use good quality filter-sterilized reagents to prevent contamination issues later.
*The competent cell solution contains cryoprotectants at optimal concentrations, and these concentrations should be standardized in your laboratory depending on the yeast strain used and the concentration of cells. A standard protocol by Gietz and Schiestl utilizes a 5% glycerol + 10% DMSO mix, and this has been used extensively in various labs worldwide for making competent cells with only slight variations or adaptations.1
It’s All Down to the Cryoprotectants
Glycerol and dimethyl sulfoxide (DMSO) are the most common cell-permeable cryoprotectants used to prepare competent cells. They penetrate cells and prevent the formation of ice crystals that could cause membrane rupture during freezing. Glycerol and DMSO are intracellular agents, so they also possess certain disadvantages. They may lead to cell toxicity, especially during the thawing process. This leads us to find other alternatives that are milder on cells and do not compromise on cell efficiency. Non-permeable agents like sucrose and trehalose can be used.2 Sorbitol with calcium is also a good cryoprotectant. It’s especially used in protocols demanding electroporation for making competent cells.3
Storing Competent Yeast Cells
Using fresh competent cells each time you perform a transformation is advisable because transformation efficiency could decrease with longer storage of competent cells, but this isn’t always feasible.
Competent yeast cells can be stored at -80°C for up to one year without loss of transformation potential1. However, flash freezing in liquid N2 or in a -80° freezer is not recommended for competent yeast cells. Competent yeast cells need to undergo slow freezing like mammalian cells in a cryoprotectant. This can be carried out as follows:
- Use a freezing device like Mr. Frosty or a cardboard or Styrofoam box for this purpose.
- Insulate the cell vials inside the box using pieces of paper, Styrofoam peanuts or cardboard shreds.
- During recovery, thaw cells at 37°C and wash out the antifreeze agents with rich medium prior to transformation.
In the end, only a few key steps are necessary to obtain high quality competent yeast cells. If the cells are of prime health after optimal growth conditions and the reagents are of good quality, little can go wrong, so follow our tips for success transformation and great experimental results!
Do you have another tip? Share it with us by writing in the comments section!
References and Further Reading
1. Gietz RD, Schiestl RH. (2007) Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2(1):1–4.
2. Diniz-Mendes L, Bernardes E, de Araujo PS, Panek , Paschoalin VM. (1999) Preservation of frozen yeast cells by trehalose. Biotechnol Bioeng. 5;65(5):572–8.
3. Suga M, Isobe M, Hatakeyama T. (2000) Cryopreservation of competent intact yeast cells for efficient electroporation. Yeast. 16(10):889–96.
Leave a Comment
You must be logged in to post a comment.