Project description:This SuperSeries is composed of the following subset Series: GSE5075: Aerobic transcriptional responses of Escherichia coli to NO under defined chemostat conditions. GSE5076: Anaerobic transcriptional responses of Escherichia coli to NO under defined chemostat conditions. GSE5084: Anaerobic NO-exposed Chemostat Comparison of Wt & norR mutant Responses GSE5137: Aerobic NO-exposed Chemostat Comparison of Wt & norR mutant Responses GSE5139: Aerobic NO-exposed Chemostat Comparison of Wt & hmp mutant Responses Keywords: SuperSeries Refer to individual Series
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 54 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.1 h-1. In order to establish anaerobic growth, nitrogen was sparged through the chemostat medium prior to inoculation and throughout the course of the experiment at a rate of 0.2 l/min. No dissolved oxygen was detectable using the OxyProbe. Sodium fumarate was added to anaerobic medium at a final concentration of 50 mM to act as a terminal electron acceptor. Cells were grown as above to steady-state, At steady-state, NOC-5 and NOC-7 were added to the chemostat culture and to the nutrient feed at a final concentration of 10 uM of each, unless otherwise stated. Samples were taken immediately prior to the addition of NOCs and after a period of 5 min exposure to NOC for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagenâ??s RNeasy Mini kit as recommended by the suppliers. Equal quantities of RNA from control and NOC-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. 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). 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. Two biological repeats (ie. two chemostat runs) were grown. Control and exposed samples were taken as described in summary. For each chemostat run 2 hybridisations were performed. One in which the control was Cy3 labelled whilst the exposed was Cy5 labelled, and a dye swap.
Project description:Purpose: This study aimed to identify the genes regulated by plasmid-encoded regulator C (PerC). Whole transcriptomes of WT typical enteropathogenic E. coli (tEPEC) strains E2348/69 and coisogenic null-perC mutant JPEP22 were analyzed to identify and quantify differentially expressed genes. Methods: RNA was isolated from the WT and null-perC mutant strains and RNA integrity (RIN) was determined to be 10 out of possible 10 for all samples by Aligent Bioanalyzer. rRNA was depleted and resulting mRNA was reverse-transcribed into cDNA. Libraries were multiplexed for discrimination between libraries, barcoded for sequencing, and amplified with random hexamers for 15 PCR cycles. Transcripts were sequenced for 50 bases in single-end fashion within one lane of an Illumina Hiseq 2000 flow cell. This yielded roughly 30 million reads per sample. Results: Differential gene expression (DGE) analysis showed that 157 genes were statistically significantly regulated using a false-discovery rate (FDR) of 10% (q ≤ 0.10). Of these genes, the perC-mutant strain had far greater transcripts for the fim operon genes, fewer transcripts of nitrate reductase genes and anaerobic metabolism genes, and fewer transcripts for Hfq-dependent ncRNAs compared to WT. Conclusions: Differential transcript abundance between the perC-mutant and WT strains indicate PerC's negative regulation of the fim genes and positive regulation of anaerobic metabolism and ncRNA genes.
Project description:Investigation of whole genome gene expression level changes in a Escherichia coli MG1655 K-12 ?fnr mutant, compared to the wild-type strain. The mutations engineered into this strain produce a strain lacking the FNR protein. WT strains were grown under aerobic and anaerobic growth conditions.
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 54 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.1 h-1. In order to establish anaerobic growth, nitrogen was sparged through the chemostat medium prior to inoculation and throughout the course of the experiment at a rate of 0.2 l/min. No dissolved oxygen was detectable using the OxyProbe. Sodium fumarate was added to anaerobic medium at a final concentration of 50 mM to act as a terminal electron acceptor. Cells were grown as above to steady-state, At steady-state, NOC-5 and NOC-7 were added to the chemostat culture and to the nutrient feed at a final concentration of 10 uM of each, unless otherwise stated. Samples were taken immediately prior to the addition of NOCs and after a period of 5 min exposure to NOC for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. Equal quantities of RNA from control and NOC-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. 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). 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. Keywords: Stress Reponse, Continuous culture, Chemostat, NO, Nitric oxide
Project description:Escherichia coli strain MG1655 was grown in parallel chemostat cultures in GGM. In one chemostat the culture was fed adequate Zn; in the other, measures were taken to eliminate Zn from the chemostat vessel and culture medium. Culture volume (120 ml), temperature (37 oC) and stirring speed (437 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. After 50 hours, samples from the adequate Zn and Zn-limited chemostats were harvested into RNAprotect and total RNA was purified using Qiagen’s RNeasy Mini kit (using the supplier’s protocol) prior to use in microarray analysis. Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats.
Project description:Investigation of whole genome gene expression level changes in a Escherichia coli MG1655 K-12 ?fnr mutant, compared to the wild-type strain. The mutations engineered into this strain produce a strain lacking the FNR protein. WT strains were grown under aerobic and anaerobic growth conditions. A six chip study using total RNA recovered from two separate cultures of Escherichia coli MG1655 K-12 WT (aerobic and anaerobic) and two separate cultures of the ?fnr mutant strain (anaerobic). Each chip measures the expression level of 4,661 genes from Escherichia coli MG1655 K-12 with eight 60-mer probes per gene, with each probe represented twice on the array.