Transcriptional profile of pulsed AsxR in Escherichia coli O157:H7 str. TUV93-0
ABSTRACT: Transcript abundance in Escherichia coli O157:H7 was determined in the presence or absence of pulsed expression of the small RNA, AsxR. AsxR was cloned under the control the arabinose inducible promoter Para. Escherichia coli O157:H7 str. TUV93-0 with pAsxR or empty vector was cultured in MEM-HEPES media to an OD600 of 0.8 and 0.2% arabinose added. 10min after addition of arabinose 10ml of cells were harvested and and pellets resuspended in 1ml of Trizol and total RNA isolated. RNAs were labelled using the SuperScript Plus indirect cDNA labelling System. Triplicate control RNAs were pooled and hybridised to seperate AsxR test RNAs on three microarays. Arrays were hybridised using the Maui hybridisation platform and Scann using and Axon Autoloader Scanner. GenePix software was used to analyse images and GPR files were analysed using Genespring 7.3.1.
Project description:Escherichia coli O157 presents a number of specific problems in terms of food safety and public health. It has been found that E. coli O157 is more resistant to a number of the stresses encountered during food production such as heat, pH and osmotic shock. This greater resistance is thought to contribute to the low infectious dose of E. coli O157 (<100 organisms). Moreover, E. coli O157 is associated with debilitating conditions such as haemorrhagic colitis and haemoytic uraemic syndrome, particularly in children and the elderly. We have been studying the stress responses of E. coli O157:H7 (Sakai) and comparing with a commensal strain of E. coli K-12, MG1655. We found that E. coli O157 (Sakai) is more resistant to heat stress than MG1655. A microarray study of these strains subjected to sub-lethal heat-stress at 45°C was carried out. In E. coli O157 (Sakai), 380 genes responded significantly to the treatment compared to 410 genes in MG1655. Overnight cultures of E. coli O157 (Sakai) and E. coli K-12 MG1655 were grown in Neidhardt's EZ Rich Defined Medium and diluted 1:100 in 50 ml fresh medium in 125 ml Ehrlenmeyer flasks. The cultures were shaken at 37°C until the optical density (OD600) reached 0.4. Each culture was divided into 2 equal parts in identical flasks. One flask flask was transferred to a shaking water bath and incubated at 45°C for 10 min; the other flask was incubated at 37°C for 10 min. After incubation, the cultures were transferred to 50 mL centrifuge tubes and treated with RNAprotect™ to stabilise the mRNA. The experiment was performed 3 times on different days. Six custom-made microarray slides were used in this study; each slide was hybridised with labelled cDNA made from untreated and heated E. coli O157 (Sakai) or MG1655.
Project description:Six isolates of PT21/28 and six of PT32 were analysed by CGH using UBECarray3 microarrays (containing probes for E. coli K-12 str. MG1655 and O157:H7 str. EDL933 and Sakai) to define genotypic differences between phage types. gDNA from E.coli O157 str. Sakai was hybridised to all arrays to provide a universal control channel on all arrays. gDNA from 12 PT 21/28 & 32 isolates were labelled with Cy5 and control gDNA from str. Sakai was labelled with Cy3. Test and control gDNA was hybridised to UBECarray3 microarrays. The LOWESS normalised relative signal to the Sakai control channel was used to compare between samples.
Project description:The transcriptional profile of Escherichia coli O157 treated with small molecule inhibitors of type III secretion was determined. Four variations of the small molecule inhibitor were assessed for global changes in transcription by treating cells with 20uM of inhibitor or an equivalent volume of DMSO (inhibitor solvent). Keywords: treatment, dose, Cy3, Cy5, 2-colour For each inhibitor, biological tripicates of treated (20uM inhibitor in DMSO) and untreated (DMSO only) cultures were grown in 25ml of MEM-HEPES supplemented with 0.1% Glucose and 250nM Fe(NO3)2. RNA was extracted from 15ml of culture at OD 0.7 and labeled with Cy5 (treated) and Cy3 (untreated) (excepting ME0055 that was performed as a dye swap). Labeled untreated RNA was pooled prior to hybridisation. Treated RNA (biological replicates) and pooled untreated RNA was hybridised to microarray slides. Slides were scanned using an Axon 4100A scanner and data processed using Genespring GX.
Project description:This SuperSeries is composed of the following subset Series: GSE10295: Bj_Heterotrophy vs. Arabinose supplemented chemoautotrophy GSE10296: Bj_Heterotrophy vs. Chemoautotrophy GSE10298: Bj_Chemoautotrophy vs. Arabinose supplemented chemoautotrophy Refer to individual Series
Project description:Tpx, FolX and WrbA were identified as a targets for type 3 secretion inhibititors in pull-down assays.The transcriptional profile of Escherichia coli O157:H7 and isogenic mutants grown in MEM-HEPES were determined. Total RNA from triplicate cultures growing exponentially in MEM-HEPES media were labelled with Cy5 (wild type) and Cy3 (tpxC61S). Labelled cDNAs were hybridised to UBEC3 arrays.
Project description:Transcriptional profiling of arabinose supplemented chemoautotrophically grown cells Keywords: Comparison of different lifestyles Three independent biological materials were prepared for arabinose supplemented chemoautotrophically cultured cells and heterotrophically cultured cells. Total 6 arrays including dye swap were analyzed.
Project description:Transcriptional profiling of E.coli O157:H7 cells comparing control untreated cells with PEG8000treated cells Two-condition experiment, Control vs. PEG8000. Biological replicates: 1 control, 1 treated.
Project description:We used microarray CGH analysis with a tiling path BAC DNA microarray to profile DNA copy number alterations in 164 serous ovarian adenocarcinomas. Survival probabilities modelled by proportional hazards were used to stratify cases into good, intermediate or poor survival groups. Comparison of aCGH data from these groups was used to identify genomic alterations associated with patient survival. A total of 984 cases of serous ovarian adenocarcinoma from three different studies were combined and Cox proportional hazards used to model survival using patient age, tumour stage and residual disease status as covariates. Survival probabilities (Pr) were extracted for all cases and used to stratify 384 cases for which microarray CGH data was available. From these, we identified 70 cases with poor survival (Pr>0.784) and 70 cases with good survival (Pr<0.35). aCGH data from these cases was provided to an iterative Support Vector Machine (SVM) routine to detect copy number changes associated with survival. Candidate regions were then validated by survival analysis in all 384 cases for which aCGH data was available. This series contains aCGH data from 164 tumours from the MALOVA collection.