strain: C. glutamicum type strain ATCC 13032 sample: BHI_wild_type_II
Treatment protocol
see growth protocol
Growth protocol
For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5 by centrifugation with crushed ice (4°C; 4,000 x g; 3 min).
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott, (2004) Mol Microbiol 54: 420-438.)
Label
Cy5
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm (2003) Appl. Environm. Microbiol. 69:2521-2532 and in Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott. (2004) Mol. Microbiol. 54: 420-438.
strain: C. glutamicum delta pup sample: BHI_delta_pup_II
Treatment protocol
see growth protocol
Growth protocol
For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5 by centrifugation with crushed ice (4°C; 4,000 x g; 3 min).
Extracted molecule
total RNA
Extraction protocol
The preparation of total RNA was performed as described previously with the RNeasy Kit from Qiagen (Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott, (2004) Mol Microbiol 54: 420-438.)
Label
Cy3
Label protocol
Synthesis of fluorescently labelled cDNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm (2003) Appl. Environm. Microbiol. 69:2521-2532 and in Möker, N., M. Brocker, S. Schaffer, R. Krämer, S. Morbach & M. Bott. (2004) Mol. Microbiol. 54: 420-438.
Hybridization protocol
Purified cDNA samples to be compared were pooled and the prepared two-color samples were hybridized at 65°C while rotating for 17 hours using Agilent’s Gene Expression Hybridization Kit, hybridization oven and hybridization chamber. After hybridization the arrays were washed using Agilent’s Wash Buffer Kit according to the manufacturer’s instructions.
Scan protocol
Fluorescence of hybridized DNA microarrays was determined at 532 nm (Cy3) and 635 nm (Cy5) at 5 μm resolution with a GenePix 4000B laser scanner and GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). Fluorescence images were saved to raw data files in TIFF format (GenePix Pro 6.0). Quantitative TIFF image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0).
Data processing
For background correction of spot intensities, ratio calculation and ratio normalization, GPR-files were processed using the BioConductor R-packages limma and marray (http://www.bioconductor.org).
