E. coli samples for RNA extraction were taken during fermentation. Cells were harvested by centrifugation at the cultivation temperature (37°C, 10,000 g, 1 min), separated from the supernatant, and rapidly frozen in dry ice. The samples were stored at -70°C until analysis
Growth protocol
All cultures were carried out in a 5 l fermentor BIOSTAT B-DCU (Sartorius BBI Systems Inc. Melsungen) at controlled temperature 37°C, pH 7.0, and dissolved oxygen tension 30%. The culture volume was 2 l. All reagents were purchased from Sigma-Aldrich, Inc. The synthetic culture medium had the following composition (g l-1): 8, glucose; 14.6, K2HPO4; 3.6, NaH2PO4·H2O; 2.68, (NH4)2SO4; 2, Na2SO4; 1, MgSO4; 1, Na-citrate; 0.5, NH4Cl; 2 ml of 10 mg l-1 of thiamine; 3 ml of trace element solution (g l-1 20.0, Na-EDTA; 15.0, FeCl3·6H2O; 0.5, CaCl2·2H2O; 0.2, ZnSO4·7H2O; 0.2, CoCl2·6H2O; 0.2, CuSO4·5H2O; 0.2, MnSO4·4H2O). The chemostat culture was performed with adding feed medium
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions
Label
biotin
Label protocol
Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 6 microgram total RNA (Expression Analysis Technical Manual, 2001, Affymetrix)
Hybridization protocol
10 microgram of cDNA were hybridized for 16 hr at 45C on GeneChip E. coli Antisense Genome Array (Affymetrix, USA) chips
Scan protocol
GeneChips were scanned with DNA Microarray Scanner BA (Agilent Technologies, Inc, CA)
Description
BL21/pOri2 (mu=0.20)
Data processing
Expression data were analyzed with the Microarray Analysis Suite, version 5.0 (Affymetrix). Global scaling was performed to compare genes between chips. Each chip was normalized to a target intensity value of 2,500. Expression analysis files created by Microarray Suite, version 5.0, were exported to Microsoft Excel for data formatting.
