Identification of yeast genes involved in Sauvignon Blanc aroma ...

34 MATERIALS AND METHODSx 10 6 cells/mL in a 100-well microtiter plate, which was then covered with a tight fittingplastic lid. After placing the microtiter plate into the temperature controlled BioscreenC analyzer, OD readings of up to 200 samples (two plates) were taken simultaneouslyevery 15 min. Cultures were shaken vigorously (setting high) for 75 s prior to each measurement.All experiments were initially carried out in quintuples and were reduced totriplicates by omitting the samples with the lowest and highest OD readings after the experiment.Negative uninoculated controls were always included, but are not shown in theresults.2.6 Yeast growth assays for cysteinylated derivativesThis assay was developed to determine if yeast is able to use cysteinylated derivatives asa sole sulfur and nitrogen source. The development of the assay is described in furtherdetail in Appendix A.1.2.6.1 Cysteinylated derivatives as a sole sulfur sourceThe sulfur-free MSD medium for yeast is based on Cherest and Surdin-Kerjan (1992) andis listed in Table 2.3. Single yeast colonies were transferred into a flat-bottomed, 96-wellmicrotiter plate filled with 200 µL of YPD-broth per well, sealed with sticky aluminiumfoil and grown overnight at 28 ◦ C and 200 rpm shaking. By using a 96-pin replica-plater,pre-cultured cells were picked up off the plate, dipped two times into 96-well-plates filledwith sterile water to get rid of the YPD-broth and to dilute the cells, before the starvationmedium consisting of sulfur-free MSD medium was inoculated. After incubation for24 h as described above, cells were then replica plated into sulfur-free medium supplementedwith the appropriate cysteinylated derivative at a final concentration of 0.5 mMand incubated for 48 h as described above. The addition of cysteine (0.5 mM) was usedas a positive control and the medium without any sulfur as negative control. Growth wasscored by assessing final cell density visually on a scale of 0-4.2.6.2 Cysteinylated derivatives as a sole nitrogen sourceCysteinylated derivatives were also tested if they could be used by yeast as a sole nitrogensource. The procedure was followed as described in section 2.6.1 with the exception