Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 265

3.3 Rapid Yeast
Transformation

This procedure is faster and simpler than Subheading 3.2 but gives
signifi cantly lower yields—use only for test-mode experiments if
enough plasmid DNA is available. The following recipe is for one
transformation; scale up as necessary and divide samples from step
4 onwards.

1. Grow cells in 3 ml YEPD overnight at 28 °C with shaking.
2. Centrifuge at 2,500 × g for 7 min ( see Note 11 ) and remove
supernatant.
3. Wash with 3 ml of sterile water and spin down at 2,500 × g for
7 min; remove supernatant.
4. If doing multiple transformations from a scaled-up culture,
divide the suspension into aliquots. To each aliquot, add
100 g

plasmid DNA in minimal volume. It is recommendable to
include also mock transformation without plasmid DNA as a
control for absence of contamination.
5. Vortex the mixture briefl y and add 500

μ g denatured single-stranded carrier DNA and 1-2 μ

l PEG/LiAc mix.
Incubate at room temperature with gentle mixing (300 rpm
on a vortex shaker) for 15 min.
6. Incubate at 42 °C for 10 min.
7. Add 96 % ethanol to a fi nal concentration of 10 % and con-
tinue incubation for another 5 min.
8. Pellet cells at 2,500 × g , 5 min. Wash cells with sterile water
and centrifuge again as before.
9. Resuspend cells in 200

μ

l of sterile water and plate onto appro-
priate selective agar medium. Select for the presence of plas-
mid only, i.e., incubate the plates at a permissive temperature
afterwards if working with temperature-sensitive strains.

μ

3.4 Evaluating
Effects of
Heterologous Protein
Expression:
Experiment Design

When studying the effects of heterologous gene expression in
yeast, always include appropriate controls.

1. As a minimum, compare two identically treated cultures, ide-
ally with several biological replicates such as independent
transformed clones: (a) a transformant carrying and expressing
your gene of interest (i.e., a complemented mutant or a wild-
type strain with an overexpression phenotype) and (b) an
empty vector transformant, carrying the plasmid used for
overexpression but without your gene of interest. In case of
complementing a mutation causing a conditional growth
defect, add (c) also an isogenic wild-type strain, preferentially
also transformed with the empty vector ( see Note 12 ).
2. For additional verifi cation, you may also include (d) deriva-
tives of the original transformant devoid of the plasmid
( see Subheading 3.6 ) together with (e) the original