genotype: Tol1 growth phase: Early exponential phase of growth
Treatment protocol
Methanol was added from the beginning of the growth.
Growth protocol
C. glutamicum and C. glutamicum Tol1 were cultured in LB medium for overnight and inoculated into 50 mL LB complex medium or 50 mL mCGXII medium with 100 mM of glucose at the initial O.D. of 1. Cells were harvested in the early exponential growth phase (O.D. between 6 and 8).
Extracted molecule
total RNA
Extraction protocol
RNA isolation was performed as described previously with some modification (Wendisch, V. F. (2003). Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. J. Biotechnol. 104, 273–285.). Total RNA was isolated using the RNeasy Mini Kit(Qiagen). The purified RNA was analyzed by spectrophotometer (NanoDrop) for quantity and gel electrophoresis for quality. The RNA sample was stored at −80 °C until further use.
Label
Cy5
Label protocol
cDNA synthesis from total RNA and labeling were performed as described previously (Netzer, R., Krause, M., Rittmann, D., Peters-Wendisch, P. G., Eggeling, L., Wendisch, V. F., and Sahm, H. (2004). Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch. Microbiol. 182, 354–363.; Polen, T., Schluesener, D., Poetsch, A., Bott, M., and Wendisch, V. F. (2007). Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol. Lett. 273, 109–119. ).
genotype: Tol1 growth phase: Early exponential phase of growth
Treatment protocol
Methanol was added from the beginning of the growth.
Growth protocol
C. glutamicum and C. glutamicum Tol1 were cultured in LB medium for overnight and inoculated into 50 mL LB complex medium or 50 mL mCGXII medium with 100 mM of glucose at the initial O.D. of 1. Cells were harvested in the early exponential growth phase (O.D. between 6 and 8).
Extracted molecule
total RNA
Extraction protocol
RNA isolation was performed as described previously with some modification (Wendisch, V. F. (2003). Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. J. Biotechnol. 104, 273–285.). Total RNA was isolated using the RNeasy Mini Kit(Qiagen). The purified RNA was analyzed by spectrophotometer (NanoDrop) for quantity and gel electrophoresis for quality. The RNA sample was stored at −80 °C until further use.
Label
Cy3
Label protocol
cDNA synthesis from total RNA and labeling were performed as described previously (Netzer, R., Krause, M., Rittmann, D., Peters-Wendisch, P. G., Eggeling, L., Wendisch, V. F., and Sahm, H. (2004). Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch. Microbiol. 182, 354–363.; Polen, T., Schluesener, D., Poetsch, A., Bott, M., and Wendisch, V. F. (2007). Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol. Lett. 273, 109–119. ).
Hybridization protocol
DNA microarray hybridization was performed as described previously with modification (Netzer, R., Krause, M., Rittmann, D., Peters-Wendisch, P. G., Eggeling, L., Wendisch, V. F., and Sahm, H. (2004). Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch. Microbiol. 182, 354–363.; Polen, T., Schluesener, D., Poetsch, A., Bott, M., and Wendisch, V. F. (2007). Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis. FEMS Microbiol. Lett. 273, 109–119. ). Hybridization and washing was performed with HS4800 (Tecan).
Scan protocol
Image was attained with LS200 (Tecan).
Description
Cultivation in mCGXII minimal medium 100 mM glucose compared to growth with additional 120 mM methanol
Data processing
Normalization and evaluation of the microarray data was performed with the software package EMMA 2 (Dondrup, M., Albaum, S. P., Griebel, T., Henckel, K., Jünemann, S., Kahlke, T., Kleindt, C. K., Küster, H., Linke, B., Mertens, D., et al. (2009). EMMA 2--a MAGE-compliant system for the collaborative analysis and integration of microarray data. BMC Bioinformatics 10, 50.).
