Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 267

3.6 Verifi cation
of Complementants
by Plasmid Loss

To make sure that the observed phenotype(s) are caused by the
plasmid carrying the heterologous gene rather than a modifi er
mutation ( see Note 10 ), verify that your transformant reverts to the
phenotype of the original host strain after evicting the plasmid.

1. Grow a fresh YEPD plate culture of the transformed yeast
strain at permissive temperature until nice colonies develop
(approx. 2-3 days). Alternatively grow a 2 ml culture to sta-
tionary phase (about 2 days with shaking) in liquid YEPD.
2. If the URA3 marker was used for plasmid selection, use the
blunt end of a sterile toothpick to transfer a pinhead-sized
amount of biomass from the YEPD plate from step 1 to a
5-FOA plate and spread it to the size of a thumbnail. Alternatively,
centrifuge 1 ml of the liquid culture from step 1 in an Eppendorf
tube, resuspend in 1 ml of sterile water, and plate 0.1 ml on a
5-FOA plate. Incubate at a permissive temperature. Colonies
formed by plasmid-free cells will develop in a couple of days.
Pick them with the sharp end of a toothpick and re-streak on a
YEPD plate to obtain more material. Verify that they are ura-
by parallel streaking to +URA and −URA plates.

3. If you used any other marker, scratch the biomass off the
YEPD plate from step 1 or wash it off with sterile water to
obtain a cell suspension. Dilute this suspension, or the liquid
culture from step 1 , into 1 ml of water to a slightly cloudy
appearance and determine cell count using a hemocytometer
(the counting step may be skipped once you acquire experi-
ence). Brief sonication may help to break cell clumps.
4. Prepare serial dilutions in sterile water to achieve 500-1,000
and 100-250 of cells per 0.2 ml. Spread 0.1 and 0.2 ml ali-
quots of each dilution on YEPD plates and incubate at permis-
sive temperature till colonies develop.
5. Select plates with well-developed, mutually separated colonies
and replica-plate those in parallel on appropriate complete SD
and “minus” dropout plates. Alternatively, transfer about 100
well-separated colonies in parallel to complete SD and “minus”
plates using sterile toothpicks ( see Note 14 ). Pick colonies that
only grow on the complete medium and re-streak them on a
YEPD plate to obtain more material.
6. Compare the relevant growth-related and morphological
phenotype(s) of the resulting plasmid-free segregants with the
original transformant and the non-transformed host strain.

1. Grow cells in 3 ml of selective media (e.g., SD-Ura for an
URA3 -containing plasmid) overnight at 28 °C with shaking.
2. Pellet cells at maximum speed for 2 min ( see Note 11 ). Discard
supernatant.
3. Add 180

3.7 Plasmid Rescue
from Yeast

μ l of GTE buffer, resuspend, and add 100 μ l of acid-

washed glass beads. Vortex at full speed for 5 min.