The advantage of using yeast for transformation instead of other eukaryotic organisms is that they are less expensive, easier to grow, easier to manipulate and we can obtain a higher protein yield. However, some post-transcriptional modification are not similar to other eukaryotic systems and protein may not have the correct tertiary structure.

 

We will be focusing on transformation methods for Saccharomyces cerevisiae because it is the yeast most used for transformation methods. However, P. pastoris is also a common yeast for transformation that has a higher protein expression and secretes lower native proteins.Therefore, the protein secreted is going to be mostly recombinant protein of interest that will be more pure. The research says that DNA enters the cell via endocytosis but how it reaches the nucleus is unknown. Although S. pombe, C. albicans, P. pastoris and Aspergillus are also used.There are many methods for transformation like spheroplast method, lithium method, electroporation, biolistic method and glass bead method being the most important ones. The transformation efficiency with electroporation is very high but the most common method is lithium acetate. [2] [3] 

 

Spheroplast method [2]:

The cell wall of the yeast is removed with enzymes (resulting in relaxation of shape and a spherical appearance) and DNA is taken up in the presence of CaCl2 and polyethylene glycol (PEG). Takes a while and the cells are very fragile after transformation.This method is not very popular nowadays due to the complex procedure. Moreover, the transformants are embedded in regeneration agar hindering replica plating. However, is still used with yeast artificial chromosomes that have 100-1000kb size and with infectious prion particles. Usually lithium acetate. Treatment with monovalent alkali cations (Li+) can be induce cells to take up DNA in the presence of PEG and followed by brief heat shock of cells.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                             Figure.1 Steps in yeast transformation by sphereplast method.

Lithium method [2]:

This method was first published in 1983. They decided to use lithium because is it known to be effective in eluting inorganic polyphosphate, a negatively charged macromolecule like DNA. They established that monovalent cations like Li help in the transformation efficiency because of their mild chaotropic effect. LiAc is more efficient than LiCl. It also help incubating the cells in polyethylene glycol (PEG) and plasmid DNA, the heat-shock enhances the efficiency and the cells being in mid-log phase. 2-mercaptoetanol, dithiothreitol, DMSO or ethanol can also be added to improve the efficiency.

Electroporation [4]:

Perform electroporation and plate them in selective medium. The best conditions for yeast electroporation are cells in log-phase, 0.1ug of plasmid DNA for high transformation efficiency and DNA carrier is not needed. Later, Sorbitol has been included in the protocol to improve efficiency. Pre-Incubation in LiAc also improves the transformants quantity.Use of electrical current to force DNA into cells. Resuspend cells in small volume. Prepare to electroporation and is very important the cells concentration.This method has also been used in C.albicans.

 

 

 

 

 

 

 

 

 

 

 

                                                                 Figure.2 ECM 630 Exponential Decay Wave Electroporation System

 

Biolistic method:

Yeast cells can be transformed with DNA-coated metal microprojectiles that are shot into cells. This technique was first developed for transformation of plant cells and since then it is also used in bacteria, fungi, and mammalian cells. One of the advantages is that since 10-30 active plasmids are carried on each microprojectile, it is easy to introduce multiple plasmids into a single cell [32]. 

Glass bead method [5]:

Grow cells in YPD medium. Then collected by centrifugation, wash twice with SD medium made 1 M sorbitol, and resuspended in SD, 1 M sorbitol at 1/10 the original culture volume. Aliquots of 0.2 ml will be placed in 15-ml plastic tubes with 5 pg of plasmid DNA in 2.5 PI. Carrier DNA is 50 pg of sonicated calf thymus DNA (Pharmacia) in SD, 1 M sorbitol. Aliquots of 0.3 g of glass beads (0.45-0.52 mm diameter; Thomas Scientific) that had been weighed into glass tubes and sterilized by baking overnight at 400" were then added to tubes. The tubes containing washed cells, DNA and glass beads were mixed for 30 sec using a VortexGenie mixer (Fisher Scientific) at the highest speed. Immediately after mixing was completed, as much of the supernatant as possible was removed from the glass beads and spread on selective medium plates (SD supplemented with nutritional requirements) containing 1 M sorbitol.

 

Overall, transformation method involves three main steps:

• Preparing competent yeast cells

• Transformation with plasmid DNA

• Subsequent plating to select the transformants

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Gietz, R. D., and R. A. Woods. p824 Table.4 Advantages and Disadvantages of Transformation Methods [2]