Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 266

pre-transformation strain. For rechecking a plasmid isolated
from a library screen, compare the minimum set of strains (a)
and (b) with (f) a retransformed host strain harboring the plas-
mid isolated from the primary transformant ( see Subheading 3.7 ).
3. For conditional defects (such as temperature-sensitive growth)
grow all cultures in parallel at permissive and restrictive condi-
tions. Similarly, when using an inducible expression system,
compare inducing and non-inducing conditions.
4. Evaluate fi rst the effects of heterologous protein expression on
gross growth of yeast cultures on agar media ( see
Subheading 3.5 ). Depending on the result, progress towards
other methods such as more detailed quantitative growth eval-
uation or microscopic studies. Protocols for visualization of
various cell components in the budding yeast are available,
e.g., in ref. [ 13 ].

3.5 Drop Test for
Evaluating Effects of
Heterologous Protein
Expression on Yeast
Growth

While streaking cultures onto appropriate plates (as for colony iso-
lation) may in some cases provide initial insight into the effects on
growth, more accurate information is obtained using a drop test:

1. Grow fresh plate cultures of the strains to be tested on media
selective for plasmid retention (−URA or SD dropout) but
otherwise as gentle as possible (i.e., permissive temperature
for temperature-sensitive strains, non-inducing conditions for
inducible gene expression).
2. If (and only if) your strains grow very slowly and yield little
biomass under selective conditions, transfer a fairly massive
inoculum from the selective plate onto YEPD agar using the
blunt end of a toothpick and grow for a day or two.
3. For each strain, harvest a pinhead-size amount of biomass
using the blunt end of a toothpick and resuspend in 1 ml of
sterile water in an Eppendorf tube. Vortex well. Take a sample
of this suspension and measure its optical density (OD 550 ) after
20× dilution. Adjust all samples to the same OD using sterile
water.
4. Prepare a dilution series in sterile water with a 30× factor (i.e.,
30×, 900×, and 27,000× dilution) for each strain.
5. Spot 10 l droplets in an array of strains vs. concentrations

onto nonselective plates ( see Note 13 ), incubate at appropri-
ate conditions until colonies develop, and document photo-
graphically. The number and kind of plates depends on
experimental setup—e.g., for complementation of a tempera-
ture-sensitive defect by a galactose-regulated construct, plant
two YEPD and two YEPGal plates and incubate one plate of
each kind at the permissive and the other at the restrictive
temperature.

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