|
| Status |
Public on Feb 03, 2020 |
| Title |
Set2_WT_AmyE_ind_vs_unind_III |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT AmyE
|
| Organism |
Corynebacterium glutamicum ATCC 13032 |
| Characteristics |
genotype: WT
treatment: induced
|
| Treatment protocol |
see growth protocol
|
| Growth protocol |
Precultures of the respective C. glutamicum strains in CgXII medium containing 4% glucose were grown to an OD600 of 5-6 and used to inoculate the main cultures in the same medium to an OD600 of 0.5, which subsequently were grown to the mid-exponential growth phase. The main cultures were then split into two cultures and IPTG was added to a final concentration of 1 mM to one of the cultures (induced samples). The cells were harvested 30 min after IPTG addition by pouring the cultures into ice-containing tubes precooled to -20°C followed by centifugation (3 min, 4200 x g, 4°C). Control cultures (uninduced) did not contain IPTG. The cell pellets were quickly frozen in liquid nitrogen and stored at -80°C until use for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Preparation of total RNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm. 2003. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl. Environm. Microbiol. 69:2521-2532
|
| 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. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl. Environm. Microbiol. 69:2521-2532
|
| |
|
| Channel 2 |
| Source name |
WT AmyE
|
| Organism |
Corynebacterium glutamicum ATCC 13032 |
| Characteristics |
genotype: WT
treatment: uninduced
|
| Treatment protocol |
see growth protocol
|
| Growth protocol |
Precultures of the respective C. glutamicum strains in CgXII medium containing 4% glucose were grown to an OD600 of 5-6 and used to inoculate the main cultures in the same medium to an OD600 of 0.5, which subsequently were grown to the mid-exponential growth phase. The main cultures were then split into two cultures and IPTG was added to a final concentration of 1 mM to one of the cultures (induced samples). The cells were harvested 30 min after IPTG addition by pouring the cultures into ice-containing tubes precooled to -20°C followed by centifugation (3 min, 4200 x g, 4°C). Control cultures (uninduced) did not contain IPTG. The cell pellets were quickly frozen in liquid nitrogen and stored at -80°C until use for RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Preparation of total RNA were carried out as described in Lange, C., D. Rittmann, V. F. Wendisch, M. Bott, and H. Sahm. 2003. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl. Environm. Microbiol. 69:2521-2532
|
| 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. Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl. Environm. Microbiol. 69:2521-2532
|
| |
|
| |
| Hybridization protocol |
Custom-made whole-genome DNA microarrays for C. glutamicum ATCC 13032 printed with 70-mer oligonucleotides were obtained from Operon (Cologne, Germany) and are based on the genome sequence entry NC_006958. Hybridisation and stringent washing of the microarrays were performed according to the instructions of the supplier. Hybridisation was carried out for 16 - 18 h at 42°C using a MAUI hybridization system (BioMicro Systems, Salt Lake City, USA).
|
| Scan protocol |
After washing the microarrays were dried by centrifugation (5 min, 1600 g) and fluorescence was determined at 532 nm (Cy3-dUTP) and 635 nm (Cy5-dUTP) with 10 µm resolution using an Axon GenePix 6000 laser scanner (Axon Instruments, Sunnyvale, U.S.A).
|
| Data processing |
Quantitative image analysis was carried out using GenePix image analysis software and results were saved as GPR-file (GenePix Pro 6.0, Axon Instruments). For data normalization, GPR-files were processed using the BioConductor/R-packages limma and marray (www.bioconductor.org). Processed and normalized data as well as experimental details (according to MIAME) were stored in the in-house microarray database for further analysis.
|
| |
|
| Submission date |
Nov 20, 2019 |
| Last update date |
Apr 14, 2021 |
| Contact name |
Tino Polen |
| E-mail(s) |
t.polen@fz-juelich.de
|
| Organization name |
Forschungszentrum Jülich GmbH
|
| Department |
IBG-1: Biotechnology
|
| Street address |
Leo Brandt Str.
|
| City |
Juelich |
| State/province |
NRW |
| ZIP/Postal code |
52425 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9860 |
| Series (1) |
| GSE140735 |
The transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins |
|
| Relations |
| Reanalyzed by |
GSM5197225 |
