After inoculation from overnight cultures, E. coli cells were grown in LB medium plus 0.8% glucose at at 30C with shaking at 250 rpm in 50 ml flasks until OD 1.0.
Extracted molecule
total RNA
Extraction protocol
Extraction of total RNA was performed using Qiagen Rneasy Mini Kit according to the manufacturer's instructions.
Label
biotin
Label protocol
The Enzo BioArray terminal labeling kit with biotin-ddUTP (Affymetrix, Inc., Santa Clara, CA) was used to label the 3' termini of the fragmented cDNA.
Hybridization protocol
Hybridization solution mix was made with the labeled cDNA according to the manufacturer's instructions (Affymetrix, Inc., Santa Clara, CA), and the mixture was hybridized to the E. coli antisense genome arrays at 45°C for 16 h. A GeneChip fluidics station (Affymetrix, Inc., Santa Clara, CA) was used to automate the washing and staining of the arrays. Sequentially, the arrays were stained with ImmunoPure streptavidin (Pierce Biotechnology, Inc., Rockford, IL), antistreptavidin goat antibody (Vector Laboratories, Inc., Burlingame, CA), and R-phycoerythrin streptavidin (Molecular Probes, Inc., Eugene, OR).
Scan protocol
The probe arrays were scanned using the Affymetrix GeneArray scanner.
Description
Gene expression data for W3110 luxS mutant grown in LB plus 0.8% glucose at OD 1.0
Data processing
Microarray data were analyzed with the Affymetrix Microarray Suite software version 5.1 (Affymetrix, Inc., Santa Clara, CA) and the four-comparison survival method (7). The fluorescence of each array was normalized by scaling total chip fluorescence intensities to a common value of 500. For each growth condition, two independent experimental cell cultures (wild type) were compared with two independent control groups ( luxS mutant), and four comparisons were made. The fold change for each gene was calculated as the ratio of signal intensity for the wild type to the signal intensity for the luxS mutant. The reported value for the fold change is the average of the four comparisons. Genes with a consistent increase or decrease in all comparisons were determined and used for the analysis.
