BioProject

We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. More...

We determined the global gene expression profiles of wildtype, dam, dam mutS, and mutS mutant E. coli strains. Keywords: Basal gene expression comparison Overall design: Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further. Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin) in a parallel study (Robbins et al., unpublished results). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays. Less...