For expression measurements, samples (0.5 ml) were taken every 4 min through an interval of 200 min. The cells were centrifuged, the supernatant was decanted, and the pellet was placed in a dry-ice acetone bath. The time from removal of the sample to freezing was less than 1 min. Samples were stored at -80°C
Growth protocol
The fermenter was inoculated with 2 x 10^7 cells of the strain IFO0233 in 650 ml. Fermentative growth on glucose was observed during the first 12 h after inoculation. Oscillatory dynamics typically appear beginning 16 to 24 h after inoculation. To maintain oscillations, cultures are shifted from batch to continuous culture by medium addition and removal at a rate of 0.086/h. Once established, oscillatory dynamics remain largely unchanged for weeks to months. Normally, periodicity remains between 40 and 45 min. Dissolved oxygen levels and carbon dioxide release are the most accessible output from the oscillator and are characterized by a phase of high respiration followed by a shift to a low respiration phase. No difference in oscillation was seen in light or darkness. The oscillation is dependent on pH, aeration, and carbon dioxide. Oscillation occurs when glucose, ethanol, or acetaldehyde is used as a carbon source. Cell numbers are kept between 0.45 and 0.85 x 10^9/ml.
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, the pellet was resuspended in 0.5 ml of RNA Later (cat. no. 7020; Ambion, Austin, TX ) containing 1% 2-mercaptoethanol. Cells were lysed by beating in a minibead beater (BioSpec Products, Bartlesville, OK) for 3 min with 0.5 ml of acid-washed glass beads (cat. no. G-8772; Sigma, St. Louis, MO). After the cell lysate was removed, the beads were washed three times with 0.5 ml of RLT buffer (Qiagen, Valencia, CA) containing 1% 2-mercaptoethanol by bead beater (1 min each wash). The cell lysate and washes were pooled. An equal volume of 70% ethanol was added, and RNA was purified with RNA easy columns (Qiagen) according to the manufacturer. DNA was digested on the columns according to the instructions. RNA was eluted in a total of 0.1 ml of RNase-free water. The final RNA samples were analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Typical total RNA yields were 20-40 mg with absorbance 260/280 ratios of 1.8-2.2.
Label
Biotin
Label protocol
Purified total RNA samples were processed as recommended in the Affymetrix GeneChip Expression Analysis Technical Manual. RNA samples were adjusted to a final concentration of 1 mg/ml. Typically, 25-250 ng were loaded onto an RNA Lab-On-A-Chip (Caliper Technologies Corp., Mountain View, CA) and analyzed in an Agilent Bioanalyzer 2100. Double-stranded cDNA was synthesized from 5 mg of total RNA using GeneChip Expression 3'-Amplification Reagents One-Cycle cDNA Synthesis Kit (Affymetrix) and oligo(dT) primers containing a T7 RNA polymerase promoter. Double-stranded cDNA was used as a template to generate biotinylated cRNA using the GeneChip Expression 3'-Amplification Reagents for IVT Labeling. The biotin-labeled cRNA was fragmented to 35-200 bases following the Affymetrix protocol. Five micrograms of fragmented cRNA was hybridized to Yeast 2.0 Affymetrix arrays at 45°C for 16 h in an Affymetrix GeneChip Hybridization Oven 640. The GeneChip arrays were washed and then stained with streptavidin-phycoerythrin (SAPE) on an Affymetrix Fluidics Station 450, followed by scanning on an Affymetrix GeneArray scanner.
Hybridization protocol
Standard Affymetrix protocol
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner M10.
Description
16.1002857142857 hours following inoculation
Data processing
Results were quantified and analyzed using MicroArray Suite 6.1 and GCOS software. Matrix files have already been adjusted for differences in hybridization efficiency using a polynomial fit to the E. coli standards. This file can be obtained from the contact person, R. Klevecz.
The values for the E. coli and B. subtilis standards in the matrix file should NOT be used for any further adjustments to the data. Use the original standards in the CEL files for that purpose. Values in the matrix files were not adjusted for differences in the values of the B. subtilis standards The B. subtilis standards were added at the time of RNA isolation and should NOT be used at all except as described below.
Checking for relative constancy of RNA Recovery:
To test the possibility that differences in expression level might be due to differences in recovery of mRNA, B.subtilis poly-A standards were spiked into the cell pellet at the beginning of the RNA isolation procedure as a measure of recovery. This approach, while imperfect, gives at least some assurance that variations in total transcript levels for all transcripts in any one chip is not due to differences in recovery. It might also monitor the inherent bias in adjusting the input RNA to a constant level throughout the procedure as required by Affymetrix. No consistent trend in recovery was noted.
