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Status |
Public on Jun 28, 2011 |
Title |
Ground Sample - biological replicate 3 |
Sample type |
RNA |
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Source name |
Ground sample - control
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Organism |
Pseudomonas aeruginosa PAO1 |
Characteristics |
strain: PAO1 culture: bacteria cultured in normal ground conditions atcc: ATCC 15692
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Biomaterial provider |
American Type Culture Collection (ATCC 15692)
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Treatment protocol |
A derivative of the wild type P. aeruginosa PAO1 (ATCC 15692) was used for the spaceflight experiment, which contained a gentamicin resistance cassette. The gentamicin-resistant strain was constructed through homologous recombination as described previously (Schweizer, 1993). P. aeruginosa PAO1 was grown in LB medium containing 25 µg/ml gentamicin in the spaceflight hardware (see below) to avoid growth of any contaminants. The bacterial inoculum 1.5 x 108 CFU/ml in the spaceflight hardware was suspended in 0.5 ml phosphate buffered saline (PBS) (Invitrogen), and remained viable but -static during launch and until nine days into the flight. Here after, growth was initiated by the addition of LB as described below. Cells were fixed in-flight using the RNA and protein fixative RNA Later II (Ambion). At 2.5 hours after Shuttle landing at the Kennedy Space Center (KSC), samples were recovered and subsequently used for whole genome transcriptional microarray and proteomic analysis. In each case, the flight culture samples were compared with culture samples grown under identical conditions on the ground at KSC using coordinated activation and termination times (by means of real-time communications with the Shuttle astronauts) in an insulated room that maintained identical temperature and humidity levels as those on the Shuttle (Orbital Environment Simulator).
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Growth protocol |
P. aeruginosa PAO1 was grown in LB medium containing 25 µg/ml gentamicin in the spaceflight hardware (see below) to avoid growth of any contaminants. The bacterial inoculum 1.5 x 10+8 CFU/ml was suspended in 0.5 ml phosphate buffered saline (PBS)
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using the RNeasy minikit (Qiagen) per the manufacturer’s instructions.
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Label |
biotin
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Label protocol |
Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression protocol. Briefly, random hexamers (Invitrogen) were added to 10 microgram of RNA along with in vitro-transcribed Bacillus subtilis control spikes. cDNA was synthesized using Superscript III (Invitrogen) and the following conditions: 25°C for 10 min, 37°C for 60 min, and 70°C for 10 min. RNA was removed by alkaline treatment and subsequent neutralization. The cDNA was purified by using a QIAquick PCR purification kit (QIAGEN) and eluted in 40 microl of elution buffer (QIAGEN). The cDNA was then fragmented by using 0.6 U DNase I (Amersham) per microg cDNA at 37°C for 10 min. The fragmented cDNA was end labeled with biotin-ddUTP by using a BioArray terminal labeling kit (Enzo) per the manufacturer’s instructions. A gel shift mobility assay was performed using NeutrAvadin (Pierce) on a 5% polyacrylamide gel stained with SYBR green (Roche) to ensure complete fragmentation and labeling.
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Hybridization protocol |
Samples were hybridized, washed, stained, and scanned as described in the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
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Scan protocol |
Samples were hybridized, washed, stained, and scanned as described in the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
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Description |
gene expression data from bacteria cultured in ground conditions
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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 spaceflight and ground control cultures, 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 |
Jun 28, 2010 |
Last update date |
Jun 29, 2011 |
Contact name |
Rob Van Houdt |
E-mail(s) |
rvhoudto@sckcen.be
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Phone |
+3214332728
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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 (1) |
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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