|
| Status |
Public on Aug 22, 2013 |
| Title |
Anaerobic lactose-limited chemostat pH6.0, 35°, mixed cullture S. cerevisiae AND Lb. bulgaricus (FM12) |
| Sample type |
RNA |
| |
|
| Source name |
Lactose-limited chemostat culture 0.1h-1
|
| Organisms |
Lactobacillus delbrueckii subsp. bulgaricus; Saccharomyces cerevisiae |
| Characteristics |
c-source: Lactose
|
| Treatment protocol |
Sampling for transcriptome analysis from pure and mixed cultures was performed as previously described, using liquid nitrogen for rapid quenching of metabolism (43). Samples were stored at -80°C in a mixture of phenol/chloroform and TEA buffer until further processing for total RNA extraction.
|
| Growth protocol |
Batch and chemostat cultures were carried out in 2-l bioreactors (Applikon, Schiedam, The Netherlands), with working volumes of 1.4 l and 1.0 l, respectively. The pH was controlled at 6.0 by automated titration with 2 M KOH. The temperature was set at 35 °C and the stirring rate was set to 800 rpm. In batch cultures, anaerobicity was maintained by sparging with pure nitrogen gas or a gas mixture (2% CO2 : 98% N2) at a flow rate of 0.5 l.min-1 (CO2 2.7 and N2 5.0, Linde Gas Benelux). In chemostat cultures, the medium reservoir was sparged with pure nitrogen gas, and the culture vessel with a 2% CO2: 98% N2 gas mixture. Cultures were equipped with Norprene tubing to prevent oxygen diffusion. In chemostats, culture volume was controlled via an electrical level sensor and the dilution rate was set to 0.10 h-1. In bioreactors, the synthetic medium was supplemented with 0.3 g l-1 of antifoam A8011 (Sigma-Aldrich, St. Louis, USA). Chemostat cultures were considered to be in steady state when, after at least six volume changes, key parameters such as culture dry weight, substrate concentration and specific CO2 production rate changed by less than 2% over 2 consecutive volume changes. For each chemostat condition (yeast only, LAB only and mixed culture) three independent culture replicates were run.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Yeast total RNA from pure and mixed cultures was recovered using phenol/chloroform extraction as previously described (43). Further processing of RNA into cDNA relies on reactions initiated by the polyA tail carried by eukaryotic mRNAs only. Separation of the reaction products using the Agilent bioanalyzer confirmed that carry-over of Lb. bulgaricus RNA was negligible, thereby demonstrating that no additional purification of yeast mRNA was required. Processing of total RNA was performed according to Affymetrix’s instructions. RNA target preparation for microarray expression analysis was carried out according the GeneChip 3' IVT Express Kit (Affymetrix, Santa Clara, USA) using 200 ng of total RNA for both pure and mixed cultures. The protocol was carried out with minor modifications, i.e the Affymetrix polyA RNA controls were excluded from the aRNA amplification protocol and the IVT reactions were incubated for 16 h at 40°C. The quality of total RNA, cDNA, cRNA, and fragmented aRNA was checked using the Agilent Bioanalyzer 2100 (Agilent Technologies).
|
| Label |
Biotin-Streptavidin
|
| Label protocol |
Labeling of Affymetrix chips was performed following the manufacturer’s instructions.
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| |
|
| Hybridization protocol |
Hybridization of Affymetrix chips was performed following the manufacturer’s instructions.
|
| Scan protocol |
Scanning of the Affymetrix Genechip® microarrays was performed with The Affymetrix scanner 3000 following Affymetrix instructions
|
| Description |
S. cerevisiae and Lb. bulgaricus anaerobic lactose-limited chemostat with a dilution rate of 0.1h-1.
|
| Data processing |
Data were processed with the GeneChip® Operating Software version 1.2 (GCOS 1.2) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each
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| |
|
| Submission date |
Apr 04, 2013 |
| Last update date |
Aug 23, 2013 |
| Contact name |
Jean-Marc Daran |
| E-mail(s) |
j.g.daran@tudelft.nl
|
| Phone |
+31 15 278 2412
|
| Organization name |
Delft University of Technology
|
| Department |
Department of Biotechnology
|
| Lab |
Kluyver centre for genomics of industrial organisms
|
| Street address |
Julianalaan 67
|
| City |
Delft |
| ZIP/Postal code |
2628BC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL90 |
| Series (1) |
| GSE45776 |
Transcriptome-based characterization of the interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat co-cultures |
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