Strain and Maintenance—This study was performed with the prototrophic laboratory strain S. cerevisiae CEN.PK113-7D (MATa) [van Dijken, J. P., Bauer, J., Brambilla, L., Duboc, P., Francois, J. M., Gancedo, C., Giuseppin, M. L. F., Heinen, J. J., Hoare, M., Lange, H. C., Madden, E. A., Niederberger, P., Nielsen, J., Parrou, J. L., Petit, T., Porro, D., Reuss, M., van Riel, N., Rizzi, M., Steensma, H. Y., Verrips, C. T., Vindelov, J., and Pronk, J. T. (2000) Enz. Microb. Technol. 26, 706–714]
Biomaterial provider
L Hazelwood
Treatment protocol
Liquid nitrogen
Growth protocol
Chemostat Cultivation—Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0.Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation. The synthetic media composition was based on that described by Verduyn (1992). The media composition for aerobic cultivations was the following: for carbon-limited cultivation, 5.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 7.5 g.liter-1 glucose and 4.5 mg.liter-1 ZnSO4•7H2O. van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959. van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714. Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412. Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517. Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792. Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274. Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726. Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.
Extracted molecule
total RNA
Extraction protocol
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was g of total RNA. The quality of total RNA, cDNA, cRNA andused, starting with 15 fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x
Label
biotin
Label protocol
EukGE-ws2v4 Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target g of total RNA. The quality of totallabelling assay was used, starting with 15 RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x
Hybridization protocol
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was g of total RNA. The quality of total RNA, cDNA, cRNA andused, starting with 15 fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x
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