Transcriptional profiling of Escherichia coli after addition of CO-RMs to aerobically growing cells
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ABSTRACT:
ORGANISM(S): Escherichia coli Escherichia coli K-12
PROVIDER: GSE12877 | GEO | 2008/12/22
SECONDARY ACCESSION(S): PRJNA114165
REPOSITORIES: GEO
Similar Datasets
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 µM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.
2008-12-22 | GSE12878 | GEO
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined aerobic media. One sample was treated with 30 µM CORM-3, the other sample was a control. The volume (30 ml), temperature (37oC) and shaking (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (aerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), and K2SO4 (15 mM). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in 250 ml side arm flasks in 30 ml of the above defined minimal media (aerobic). CORM-3 was added at a final concentration of 30 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (Klett values of 30-40) before addition of CO-RM. The total 30 ml volume was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5à First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2à SSC/0.1 % SDS, 1à SSC, 0.2à SSC and 0.01à SSC. Microarray slides were dried by centrifugation at 1500 à g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-24 | E-GEOD-12877 | biostudies-arrayexpress
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 µM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (anaerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), K2SO4 (15 mM), sodium fumarate (50 mM), Luria Broth (5%) and can amino acids (0.1%). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in mini fermenter vessels in 175 ml of the above defined minimal media (anaerobic). The vessel was continually sparged with N2 to ensure anaerobiosis. CORM-3 was added at a final concentration of 100 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (OD600 of 0.4) before addition of CO-RM. For RNA extraction, 30 ml was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5à First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2à SSC/0.1 % SDS, 1à SSC, 0.2à SSC and 0.01à SSC. Microarray slides were dried by centrifugation at 1500 à g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-24 | E-GEOD-12878 | 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 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.
2013-08-21 | E-GEOD-40811 | 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 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 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:Wild type and hemA strains of Escherichia coli MG1655 were grown under anaerobiosis in batch culture in custom-made, stirred (200 rpm) 250 ml mini-fermenter vessels at 37 °C during continuous sparging with 100 % nitrogen gas. Cells were grown in defined medium (pH 7.0) containing 0.5 % glucose as the sole and limiting source of energy and carbon. The medium was supplemented with 0.1 % casamino acids and 5 % LB to support the growth of the hemA mutant. For maintenance of the hemA mutation, 50 mg/ml kanamycin were added to cultures of the heme-deficient mutant only. CORM-3 (final concentration of 100 uM) was added to wild type and mutant cultures at an OD600 of 0.2. iCORM-3 (final concentration of 100 uM) was added to only mutant cultures at an OD600 of 0.2. Samples were taken immediately prior to the addition of CORM-3 or iCORM-3 and at consecutive time-points thereafter (10, 20, 40, 60 and 120 min) for microarray analysis. Cells were harvested directly into phenol:ethanol to stabilise RNA and total RNA was purified using Qiagen’s RNeasy Mini Kit, according to the manufacturer's instructions. Time-course experiments with samples taken immediately prior to or 10, 20, 40, 60 and 120 min after addition of 100 uM CORM-3, or 100 uM iCORM-3 for the hemA mutant only, under 100 % anaerobiosis; 2 independent biological experiments were performed for each condition with 2 technical (dye swap) repeats per biological experiment.
2015-04-01 | E-GEOD-55097 | biostudies-arrayexpress
Project description:Batch cultures of Wild-type C. jejuni NCTC 11168 were grown in 200 ml volumes of Mueller-Hinton broth in 250 ml baffled flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (10 % Oxygen, 10 % Carbon dioxide, 80 % Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 42 ºC. When mid-exponential phase was reached 0.010 mM NOC-5 & NOC-7 was added to one of the cultures. After a 15 minute exposure samples of both treated and untreated cells were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagen’s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis.
2012-05-23 | GSE38115 | GEO