Yeasts are considered model systems for eukaryotic studies as they exhibit fast growth and have dispersed cells. Moreover, replica plating and mutant isolation of yeast cells can be done with relative ease and they have a well-defined genetic system. Most significantly, yeasts have a highly versatile DNA transformation system that can be utilized effectively for protein production.
The LiAc transformation method involves three main steps: preparing competent yeast cells, transformation with plasmid DNA, and subsequent plating to select the transformants. Yeast transformants are usually selected using auxotrophic markers; hence, the appropriate synthetic dropout medium is used for screening transformants.
50 % Polyethylene glycol solution (mol wt ~36,500)
1 M Tris-HCl and 0.2 M EDTA, pH 8.0 (TE Buffer) (Catalog Number T9285)
1 M Lithium acetate, pH 7.5 (Catalog Number L4158)
1x TE-LiAc solution
10 mM Tris-HCl, pH 8.0, with 1 mM EDTA and 0.1 M lithium acetate
40 % PEG in 10 mM Tris-HCl, pH 8.0, with 1 mM EDTA and 0.1 M Lithium actetate
Note: Refer to growth protocols for plating yeasts and the following section for isolating transformants.
Yeast transformants are usually selected using the URA3 complementation method, although techniques utilizing amino acids for complementation and blue-white screening methods can also be used.
In the URA3-based selection technique; the plasmid DNA has a normal copy of the yeast URA3 gene, as well as the URA3 promoter; whereas, the yeast mutant strain lacks the URA3 allele. Thus, yeast cells transformed with the plasmid have a functional copy of the URA3 gene, which allows them to grow on Yeast Synthetic Drop-out Medium Supplements without Uracil (Catalog Number Y1501).
Figure 1.Yeast Transformation