Measuring the effects of hypoxia on transcription in Caulobacter crescentus
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ABSTRACT:
ORGANISM(S): Caulobacter vibrioides
PROVIDER: GSE21127 | GEO | 2010/06/21
SECONDARY ACCESSION(S): PRJNA126637
REPOSITORIES: GEO
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Project description:To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions. Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours. After this period, the medium was considered saturated with air and the oxygen probe was set to 100%. Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring). At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%. To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition. Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%. Hypoxic cells were then harvested for RNA isolation. Four independent biological samples are included in this study. Two batches of cells were subjected to 20 minutes of hypoxia in a bioreactor; two cell batches were highly aerated.
2010-06-21 | E-GEOD-21127 | biostudies-arrayexpress
Project description:Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in Evans media containing 2 mM glucose as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CO gas was bubbled through the culture at 0.1L/min. Samples were taken immediately prior to the addition of CO gas and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to CO gas for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.
2016-09-01 | GSE50713 | GEO
Project description:Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in Evans media containing 2 mM glucose as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CO gas was bubbled through the culture at 0.1L/min. Samples were taken immediately prior to the addition of CORM-401 and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to CORM-401 for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.
2017-05-31 | GSE71784 | GEO
Project description:Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in Evans media containing 2 mM glucose as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CO gas was bubbled through the culture at 0.1L/min. Samples were taken immediately prior to the addition of CO gas and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to CO gas for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using Qiagenâ??s RNeasy Mini kit as recommended by the suppliers. Time course experiment with samples taken immediately prior to or 2.5, 5, 10, 20, 40 or 80 minutes after addition of CO gas under either 0 or 100 % perceived aerobiosis. 2 biological repeats were performed for the aerobic condition with 2 technical (dye swap) repeats per biological repeat, and three biological repeats were perfomed for the anaerobic condition with 2 technical (dye swap) repeats per biological repeat.
2016-09-01 | E-GEOD-50713 | biostudies-arrayexpress
Project description:Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 2 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CORM-3 or iCORM-3 was added to the chemostat culture at a final concentration of 40 uM. Samples were taken immediately prior to the addition of CORM-3 or iCORM-3 and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to (i)CORM-3 for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.
2013-08-21 | GSE40811 | GEO
Project description:Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Several previous transcript-profiling experiments have compared separate anaerobic and aerobic cultures. Here, for the first time, the process of adaptation was investigated by determining changes in transcript profiles when anaerobic steady-state cultures were perturbed by the introduction of air. Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 400 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (30 mM) as the carbon source with the dilution rate being 0.2 h-1. Anaerobic cultures were maintained by sparging the chemostat with oxygen-free nitrogen (95 %) and carbon dioxide (5 %) (0.2 l min-1). The switch to aerobic conditions was achieved by sparging the culture with air (2.0 l min-1) in place of the above gas mix. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions. Keywords: time course response to air
2006-07-28 | GSE4735 | GEO
Project description:Adult zebrafish (Tübingen strain, sex not specified) at approximately 1 year of age were analysed. For experiments conducted under low oxygen conditions, nitrogen gas was bubbled through water to deplete oxygen before exposure of individual fish to the medium. Oxygen concentrations were measured using a dissolved oxygen meter (DO 6+, EUTECH instruments, Singapore). The dissolved oxygen level for hypoxia treatment was measured to be 1.20 ± 0.6 mg/l, whereas normal ambient oxygen levels were 6.6 ± 0.45 mg/l. Zebrafish were exposed to the hypoxic medium for 3 hours. Briefly, after each hypoxia trial, the animals were euthanized by hypothermic shock and then decapitated to remove the brain. Total RNA was extracted from samples mentioned above using the QIAGEN RNeasy mini kit (QIAGEN, GmbH, Hilden, Germany) and stored at −80°C before further analysis. RNA concentration was determined with a NanoVue™ UV–vis spectrophotometer (GE Healthcare Life Sciences, Fairfield, USA). RNA integrity and quality were then estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and the RNA integrity number (RIN) index was calculated for each sample. Only RNAs with a RIN number >7.0 were processed further. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array (Affymetrix Inc. Santa Clara, CA). Briefly, 300ng of total RNA derived from a single adult brain was converted to amplified sense strand cDNA using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA). The resulting sense cDNA was fragmented and Biotin end-labelled using the Affymetrix Genechip WT Terminal Labeling Kit prior to hybridisation to the array at 45 °C for 16 hours.
2016-08-04 | GSE85149 | GEO
Project description:Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 2 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbeM-BM-. electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CORM-3 or iCORM-3 was added to the chemostat culture at a final concentration of 40 uM. Samples were taken immediately prior to the addition of CORM-3 or iCORM-3 and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to (i)CORM-3 for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy Mini kit as recommended by the suppliers. Time course experiment with samples taken immediately prior to or 2.5, 5, 10, 20, 40 or 80 minutes after addition of either 40uM CORM-3 or iCORM-3 under either 0 or 100 % perceived aerobiosis. 2 biological repeats were performed for each condition with 2 technical (dye swap) repeats per biological repeat.
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Project description:Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Here, the process of adaptation was investigated by determining changes in transcript profiles when aerobic steady-state cultures were depleted of air. Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 300 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (15 mM) as the carbon source with the dilution rate being 0.2 h-1. Aerobic cultures were maintained by sparging the chemostat with air (0.4 l min-1). The switch to micro-aerobic conditions was achieved by switching off the air sparging the culture. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions. Keywords: time-course, oxygen-depletion
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Project description:Adult zebrafish (Tübingen strain, sex not specified) at approximately 1 year of age were analysed. For experiments conducted under low oxygen conditions, nitrogen gas was bubbled through water to deplete oxygen before exposure of individual fish to the medium. Oxygen concentrations were measured using a dissolved oxygen meter (DO 6+, EUTECH instruments, Singapore). The dissolved oxygen level for hypoxia treatment was measured to be 1.20 ± 0.6 mg/l, whereas normal ambient oxygen levels were 6.6 ± 0.45 mg/l. Zebrafish were exposed to the hypoxic medium for 3 hours. Briefly, after each hypoxia trial, the animals were euthanized by hypothermic shock and then decapitated to remove the brain. Total RNA was extracted from samples mentioned above using the QIAGEN RNeasy mini kit (QIAGEN, GmbH, Hilden, Germany) and stored at â??80°C before further analysis. RNA concentration was determined with a NanoVueâ?¢ UVâ??vis spectrophotometer (GE Healthcare Life Sciences, Fairfield, USA). RNA integrity and quality were then estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and the RNA integrity number (RIN) index was calculated for each sample. Only RNAs with a RIN number >7.0 were processed further. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array (Affymetrix Inc. Santa Clara, CA). Briefly, 300ng of total RNA derived from a single adult brain was converted to amplified sense strand cDNA using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA). The resulting sense cDNA was fragmented and Biotin end-labelled using the Affymetrix Genechip WT Terminal Labeling Kit prior to hybridisation to the array at 45 °C for 16 hours. Two treatments including hypoxia and normoxia were studied. Each treatment had three biological replicates (i.e. three fish exposed to hypoxia and three fish exposed to normoxia). Six samples were analysed. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array. The signal intensity of the chip was scanned using a GeneChipR Scanner 3000TG and analysed using Expression Console software (www. Affymetrix.com). CEL files were imported and intensities adjusted by RMA background correction and quantile normalization.
2016-08-04 | E-GEOD-85149 | biostudies-arrayexpress