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| Status |
Public on Jul 05, 2010 |
| Title |
RWV vertical - biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
RWV
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
strain: PAO1
culture: bacteria cultured in a RWV device in vertical position (LSMMG)
atcc: ATCC 15692
|
| Biomaterial provider |
American Type Culture Collection (ATCC 15692)
|
| Treatment protocol |
Synthecon Rotating Wall Vessel bioreactors (RWV) (50 ml or 10 ml) were filled with inoculated medium so that no headspace (i.e. no bubbles) was present. Other than for stress resistance assays, for which 10 ml capacity bioreactors were used, RWV bioreactors with a capacity of 50 ml were adopted for all experiments. Identical bioreactors were mounted in triplicate on (i) a RWV device in vertical position (LSMMG) (Cellon), (ii) a RWV device in horizontal position (NG) and (iii) the center of the inner Random Positioning Machine (RPM) frame (RG) (Fokker Space), and placed in a large humidified (70%-80% relative humidity) culture chamber, to avoid evaporation of culture medium through the gas-permeable membrane at the back of each vessel (Figure 1). A 25 r.p.m. rotation speed was adopted for the RWV cultures, while RPM-cultures were randomly rotated at 10 r.p.m. (60°/s). Bacteria were grown in the three described test conditions for 24 hours.
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| Growth protocol |
The wild type P. aeruginosa PAO1 strain (ATCC 15692) was used in this study and all cultures were grown in Lennox L Broth Base (LB) (Life Technologies) at 28 °C. An overnight shaking culture (125 r.p.m.) of P. aeruginosa in LB was washed and diluted in 0.85% NaCl solution to an OD600 of 1. This bacterial suspension was used to inoculate fresh LB medium at a final concentration of 10-4 CFU/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 24 hours of cultivation, the contents of every bioreactor was gently mixed by pipetting and divided into several aliquots. Ten millilitres of culture from each growth condition was immediately fixed with RNA Protect Reagent (Qiagen), following the manufacturer's instructions, and fixed cell pellets were frozen at -20 °C until RNA extraction. Samples were immediately exposed to different stresses. The extraction of total RNA was performed with the Total RNA Isolation System (Promega). RNA quality and quantity were assessed using the Agilent Bioanalyzer 2100 electrophoresis system and the Nanodrop ND-1000 spectrophotometer (NanoDrop technologies) respectively. cDNA synthesis and further processing for the transcriptomic analysis were performed according to the protocol of the GeneChip® manufacturer (Affymetrix). Ten micrograms of total RNA per sample was used for cDNA synthesis and a Poly-A RNA control was added to the RNA sample using the GeneChip eukaryotic poly-A control kit (Affymetrix). The synthesis of cDNA was performed using denatured total RNA (70 °C for 10 min), 13 ng/µl random hexamer primers (Roche), 0.5 U/µl SUPERase In (Ambion), 25 U/µl SuperScript II reverse transcriptase (Invitrogen), 0.5 nmol/µl deoxynucleoside triphosphates in 1x first strand buffer with 10 nmol/µl dithiothreitol. The latter mix was placed in a Thermocycler operated at 25 °C for 10 min, 37 °C for 60 min, 42 °C for 60 min and 70 °C for 10 min. The remaining RNA was degraded by treatment with 1 N NaOH, incubation for 30 min at 65 °C and subsequent neutralization with 1 N HCl. cDNA was purified using a MiniElute PCR purification kit (Qiagen), where after the concentration of cDNA was assessed using the Nanodrop ND-1000 spectrophotometer (NanoDrop technologies). Eight micrograms of cDNA was fragmented to a size range of 50-200 nucleotides with DNase I (Promega) for 10 min at 37 °C where after the enzyme was inactivated at 99 °C for 10 min. The fragmented cDNA was verified by loading a sample on a 2% agarose gel containing 0.1 %0 (v/v) GelRed (Biotium).
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| Label |
biotin
|
| Label protocol |
Fragmented cDNA was end-labelled with biotin-dUTP using the GeneChip® DNA labelling reagent (Affymetrix) and terminal deoxynucleotidyl transferase (Promega) in 1x reaction buffer (Promega) at 37 °C for 60 min. The reaction was stopped using 0.5 M EDTA.
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| |
|
| Hybridization protocol |
Biotinylated cDNA was hybridized to GeneChip® P. aeruginosa Genome arrays (Affymetrix) by incubation in a GeneChip® hybridization oven 640 at 50 °C with orbital shaking at 60 r.p.m. for 16 h in the following MES-buffer [2-(N-morpholino) ethanesulfonic acid]: 0.1 µg/µl herring sperm DNA (Promega), 0.5 µg/µl bovine serum albumine (Invitrogen), 7.8% dimethyl sulfoxide (Sigma-Aldrich), 50 mM B2 control oligo (Affymetrix) in 1x hybridization buffer (made according to instructions of Affymetrix GeneChip®). Microarray slides were washed according to the manufacturer's protocol and stained using a fluidics station 450 (Affymetrix) and applying the Midi_euk2v3_450 protocol. Briefly, the staining procedure was comprised of the following steps: (i) binding of streptavidin (Invitrogen) to the biotin-labeled hybridized cDNA, (ii) binding of a biotin-conjugated streptavidin antibody (Labconsult) and (iii) binding of R-phycoerythrin-conjugated streptavidin (Invitrogen).
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| Scan protocol |
Microarrays were scanned at the emission wavelength of 570 nm where after the data analysis took place.
|
| Description |
gene expression data from bacteria cultured in vertical RWV machine
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| Data processing |
Raw Affymetrix data were pre-processed using the Affy package in BioConductor as follows: (i) background correction based on the robust multichip average (RMA) convolution model (Irizarry et al., 2003), (ii) quantile normalization to make expression values from different arrays more comparable (Bolstad et al., 2003), (iii) summarization of multiple probe intensities for each probe set to one expression value per gene using RMA (Irizarry et al., 2003). To test for differential expression between the different simulated microgravity (LSMMG and RG) conditions and the reference condition (NG) the Bayesian adjusted t-statistics was used as implemented in the LIMMA package (version 2.5.15) (Smyth, 2004). P-values were corrected for multiple testing using the Benjamini and Hochberg’s method to control the false discovery rate (Benjamini and Hochberg, 1995). Microarray analysis was performed on all three biological replicates.
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| Submission date |
Jul 06, 2009 |
| Last update date |
Jul 06, 2009 |
| Contact name |
Rob Van Houdt |
| E-mail(s) |
rvhoudto@sckcen.be
|
| Phone |
+3214332728
|
| Organization name |
SCK-CEN
|
| Department |
Interdisciplinary Biosciences
|
| Lab |
Microbiology Unit
|
| Street address |
Boeretang 200
|
| City |
Mol |
| ZIP/Postal code |
2400 |
| Country |
Belgium |
| |
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| Platform ID |
GPL84 |
| Series (2) |
| GSE16970 |
Response of Pseudomonas aeruginosa PAO1 to low shear modeled microgravity |
| GSE22684 |
Transcriptional and proteomic response of Pseudomonas aeruginosa PAO1 to spaceflight conditions involves Hfq regulation and reveals a role for oxygen |
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