|
| Status |
Public on Aug 30, 2013 |
| Title |
Technical replicate 3 of 3, Control D. shibae aerob, Sample D. shibae 20 min anaerob |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Dinoroseobacter shibae, aerob
|
| Organism |
Dinoroseobacter shibae |
| Characteristics |
strain: DSM 16496T
growth phase: exponential phase
|
| Treatment protocol |
An anaerobic shift was performed after two reactor residence time points (20 hours) by stopping the aeration. The investigated time points were 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 120 min after oxygen supply switched off.
|
| Growth protocol |
Continuous cultivation of Dinoroseobacter shibae DFL12T was performed in Salt-Water-minimal medium (SWM) in an Infors HT Multifor 2 reactor (Infors, Bottmingen, Switzerland) at 30 °C, pH 8.0, with aeration of 0.7 l per minute and a stirring speed of 150 r.p.m. The reactor working volume was 1 l. The pH was adjusted automatically with 0.5 M HPO3 and 0.5 M NaOH. In the steady state the oxygen saturation of the culture was stable at approximately 85 %. To avoid aerobic anoxygenic photosynthesis of D. shibae during the experiment, the chemostat was covered with aluminium foil. The bioreactor was inoculated to a starting optical density of OD578 0.02 with a pre-culture grown in erlenmeyer flask in SWM at 30 °C and 200 rpm. Continuous flow of fresh medium was started after the culture reached an OD578 of 0.5. The dilution rate was 0.1 h-1 corresponding to the half-maximum growth rate of D. shibae in the exponential phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A 2 ml volume of a D. shibae culture was used for RNA isolation using RNA protect and RNeasy mini Kit (Qiagen, Hilden, Germany) according to the manufactures manual. Thereby, 1 µl of isolated RNA was qualified and quantified using the Agilent RNA 6000 Nano Kit and the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, USA).
|
| Label |
cy5
|
| Label protocol |
Two µg of isolated total cellular RNA was either labeled with Cy3 or Cy5, using the ULSTM Fluorescent Labeling Kit for Agilent arrays (Kreatech, Amsterdam, The Netherlands) according to the manufacturers manual.
|
| |
|
| Channel 2 |
| Source name |
Dinoroseobacter shibae, 20 min anaerob
|
| Organism |
Dinoroseobacter shibae |
| Characteristics |
strain: DSM 16496T
growth phase: exponential phase
|
| Treatment protocol |
An anaerobic shift was performed after two reactor residence time points (20 hours) by stopping the aeration. The investigated time points were 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 120 min after oxygen supply switched off.
|
| Growth protocol |
Continuous cultivation of Dinoroseobacter shibae DFL12T was performed in Salt-Water-minimal medium (SWM) in an Infors HT Multifor 2 reactor (Infors, Bottmingen, Switzerland) at 30 °C, pH 8.0, with aeration of 0.7 l per minute and a stirring speed of 150 r.p.m. The reactor working volume was 1 l. The pH was adjusted automatically with 0.5 M HPO3 and 0.5 M NaOH. In the steady state the oxygen saturation of the culture was stable at approximately 85 %. To avoid aerobic anoxygenic photosynthesis of D. shibae during the experiment, the chemostat was covered with aluminium foil. The bioreactor was inoculated to a starting optical density of OD578 0.02 with a pre-culture grown in erlenmeyer flask in SWM at 30 °C and 200 rpm. Continuous flow of fresh medium was started after the culture reached an OD578 of 0.5. The dilution rate was 0.1 h-1 corresponding to the half-maximum growth rate of D. shibae in the exponential phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A 2 ml volume of a D. shibae culture was used for RNA isolation using RNA protect and RNeasy mini Kit (Qiagen, Hilden, Germany) according to the manufactures manual. Thereby, 1 µl of isolated RNA was qualified and quantified using the Agilent RNA 6000 Nano Kit and the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, USA).
|
| Label |
cy3
|
| Label protocol |
Two µg of isolated total cellular RNA was either labeled with Cy3 or Cy5, using the ULSTM Fluorescent Labeling Kit for Agilent arrays (Kreatech, Amsterdam, The Netherlands) according to the manufacturers manual.
|
| |
|
| |
| Hybridization protocol |
300 ng of each labeled RNA were pooled, fragmented and hybridized according to the “two-colour microarray” protocol from Agilent.
|
| Scan protocol |
The DNA microarrays were scanned using a Agilent C scanner and the Agilents scan control 8.4.1 software as well as the feature extraction 10.7.3.1 software.
|
| Description |
Technical replicate 3 of 3, Control D. shibae aerob, Sample D. shibae 20 min anaerob
|
| Data processing |
Data processing was performed in the R environment (http://www.cran.r-project.org/) using the limma package (Smyth, 2005), BioBASE package (Miller, 1996.) and the GPLOTS package (Warnes, 2012) of the BioConductor project (http:// www.bioconductor.org/). Only genes with an absolute 2 fold logarithmic change (FC) >0.8 expression and a P-value <0.05 were considered in subsequent analyses.
|
| |
|
| Submission date |
May 29, 2013 |
| Last update date |
Aug 30, 2013 |
| Contact name |
Sebastian Walter Friedrich Laaß |
| E-mail(s) |
[email protected]
|
| Organization name |
Technische Universität Braunschweig
|
| Department |
Inst. f. Mikrobiologie
|
| Street address |
Spielmannstraße 7
|
| City |
Braunschweig |
| ZIP/Postal code |
38106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11243 |
| Series (1) |
| GSE47445 |
Adaption of Dinoroseobacter shibae to anaerobic conditions |
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