Biomedical Engineering Reference
In-Depth Information
2. Pin yeast from a 96-density solid agar array (as above) with a
sterile replicator into 96-well plates with medium containing
15 % sterile glycerol.
3. Incubate 2 days at 30 °C.
4. Freeze at −80 °C.
5. Remove the frozen arrays briefl y from freezer and apply sealing
tape, and then return to freezer in order to minimize the risk
of cross-contamination.
3.2 Invertase Assay
Invertase enzyme, encoded by the
SUC2
gene, hydrolyzes sucrose
into glucose. The glucose produced is oxidized by glucose oxidase,
which releases hydrogen peroxide as a by-product. Horseradish
peroxidase (HRP) utilizes the free peroxide to oxidize the chromo-
gen
o
-dianisidine, resulting in a change in color [
8
]. These reagents
do not penetrate the cell and so the amount of sucrose hydrolysis
depends on the amount of the GSS reporter that is located on the
surface of the cell. Therefore, mutants that alter the surface levels
of the GSS reporter can be detected [
6
].
To monitor GSS localization, sucrose hydrolysis is both initi-
ated and monitored by the addition of an agar overlay or a liquid
solution containing the necessary reagents. Glucose-containing
media will give a high background. Therefore, yeast must be grown
on media containing an alternative carbon source, such as fructose
or galactose. For the agar overlay assay, normal plasma membrane
localization results in a honey brown-colored colony, whereas
mutants with enhanced localization of GSS to the plasma mem-
brane will be much darker. In contrast, mutants that exhibit abnor-
mal localization of GSS to internal compartments will remain
white, indicating a lack of plasma membrane localized GSS (
see
Fig.
3
). These plates can be imaged with a digital camera or scanned
using a fl atbed scanner, and then analyzed by densitometry.
While the invertase overlay assay lends itself to genome-wide
analyses of yeast colony arrays, the liquid invertase assay is carried
out in multi-well plates using a plate reader and is well suited for a
medium-throughput follow-up. In the liquid invertase assay, the
normal plasma membrane localization will result in a slightly pink
color measured by the absorbance of light at 540 nm. Mutants
with enriched plasma membrane GSS will have a higher absorbance
value, whereas mutants with decreased plasma membrane GSS will
have a smaller absorbance value. To convert absorbance values to
glucose released from plasma membrane-bound GSS reporter, a
standard curve is made with known glucose concentrations.
