151_F6_G1.a
Saccharomyces cerevisiae
Protocols: Cells from culture samples were harvested rapidly by centrifugation (1 min, 4000g) and the cell pellets flash frozen in liquid nitrogen and stored at −80 °C until required. For the glucose grown chemostats, precultures of each strain were prepared by inoculating YNB minimal medium (0.67% w/v Yeast Nitrogen Base (SIGMA) and 0.5% w/v ammonium sulphate) containing 2% glucose with a single colony picked from an agar plate and incubating at 30°C 200 rpm for 24 h. Fermentors (2 L) containing F1 medium (1000 ml) with 0.25% glucose were inoculated with aliquots of the precultures to produce a starting OD600nm absorbance of 0.05, and grown in batch for 24 h (30 °C, 750 rpm stirrer speed, aeration with 1 L min−1 air). Cultures were then switched to continuous mode, maintaining the pH at 4.5 and the dilution rate at 0.11-0.12 h-1. Initial steady-state culture conditions (designated SS1) were deemed to have been established after more than five vessel volume changes and with cultures exhibiting a constant biomass, at which point culture samples were taken for analysis. For induction, DOX was added to the steady-state cultures to produce a final concentration of 5 g/ml. Culture samples were harvested for analysis at 3 (designated T1) , 6 (designated T2) and 9 h (designated T3) after induction, and a final sample taken after the cultures had reached a new steady state (SS2), as determined by the passage of six vessel volume changes and with cultures exhibiting a constant biomass. A similar protocol was followed for the acetate grown chemostat experiments except the initial preculture was performed for 42 h in YNB minimal medium containing 0.5% sodium acetate and 1% glucose. This was used to inoculate batch growth in fermentors containing YNB minimal medium with 0.5% sodium acetate and 0.05% glucose, then switched to continuous mode using only 0.5% sodium acetate as the carbon source and controlling the pH at 5.0 and the dilution rate at 0.07-0.08 h-1. After taking the induced steady state samples, designated SS2, an additional experiment was performed to analyse the response to adding glucose to the respiring, induced cultures. Glucose (40%) was added to each fermentor (and to the feed for each fermentor) to produce a final concentration of 1.6%, and culture samples taken at 15 min (designated G1), 30 min (designated G2) and 60 min (designated G3) after glucose addition. For RNA purification, frozen cells were resuspended in trizol™(Invitrogen) and lysed mechanically at 4 °C by bead beating using a FastPrep™ homogeniser (MP Biomedicals; 5×1 min cycles of shaking at 6 m/s). DNA and protein were removed from the lysed samples by extraction with chloroform (×2) and total RNA purified using RNeasy columns (Qiagen). Illumina TruSeq Stranded Total RNA Library Prep Kit
ENA first public
2018-08-23
ENA last update
2016-10-24
External Id
SAMEA4522985
INSDC center alias
University of Cambridge
INSDC center name
University of Cambridge
INSDC first public
2018-08-23T17:03:01Z
INSDC last update
2016-10-24T11:38:00Z
INSDC status
public
Submitter Id
E-MTAB-5174:151_F6_G1.a
broker name
ArrayExpress
common name
baker's yeast
genotype
TetO7pr::ybbP TDH3pr::yybT
growth condition
acetate
phenotype
DOX-inducible expression of ATP-cycling pathway
sample name
E-MTAB-5174:151_F6_G1.a
strain
BY4741-112-151