: Strain: CENPK113.7D; Genotype: MATa MAL2-8c SUC2
Biomaterial provider
Joost van den Brink
Treatment protocol
Liquid nitrogen quenching
Growth protocol
The S. cerevisiae prototrophic haploid reference strain CEN.PK113-7D (MATa) was grown at 30 °C in 2-l bioreactors with a working volume of 1.5 l as described via an electrical level sensor. Removal of effluent from the center of the culture ensured that biomass concentrations in the effluent line differed by less than 1% from those in the culture. The dilution rate was set at 0.10 h-1. The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH using an Applikon ADI 1030 Biocontroller. A stirrer speed of 800 rpm and air flow of 0.75 liter•min-1 were applied to keep the dissolved-oxygen concentration, as measured with an oxygen electrode, above 60% of air saturation in all chemostat cultivations performed. Steady-state samples were taken after 10 volume changes to avoid strain adaptation due to long-term cultivation. Biomass dry weight, metabolite, dissolved oxygen, and gas profiles were constant over at least three volume changes. Anaerobic glucose-pulse experiments were started by sparging the medium reservoir of a steady-state glucose-limited aerobic chemostat (airflow of 0.5 liter•min-1) with pure nitrogen gas. Norprene tubing and butyl septa were used to minimize oxygen diffusion into the anaerobic cultures. Two min after nitrogen sparging and just before adding the glucose, the medium pump was switched off. The 200 mM glucose pulse was injected aseptically through a rubber septum. Samples for RNA extraction were taken for each time point after the perturbation (5, 10, 30, 60 and 120 min) from two independently cultured replicates, while steady state data were taken from three independent chemostats.
Extracted molecule
total RNA
Extraction protocol
Sampling of cells from chemostats and total RNA extraction was performed as previously described in (1). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. 1- Abbott, D. A., T. A. Knijnenburg, L. M. de Poorter, M. J. Reinders, J. T. Pronk, and A. J. van Maris. 2007. Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. epub ahead
Label
Biotynylated UTP -SAPE
Label protocol
Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was used, starting with 15 mg total RNA. The quality of total RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies).
Hybridization protocol
EukGE-ws2v4 Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was used, starting with 15 mg total RNA. The quality of total RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). 1- Abbott, D. A., T. A. Knijnenburg, L. M. de Poorter, M. J. Reinders, J. T. Pronk, and A. J. van Maris. 2007. Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. epub ahead
Scan protocol
Affymetrix scanner 3000
Description
JB03
Data processing
GeneChip® Operating Software (GCOS), version 1.2. Samples scaled to 150.
