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Series GSE2999 Query DataSets for GSE2999
Status Public on Jul 27, 2005
Title Cisplatin-induced gene expression in DNA adenine methyltransferase (dam) and mismatch repair deficient E. coli
Platform organism Escherichia coli K-12
Sample organism Escherichia coli
Experiment type Expression profiling by array
Summary We used Affymetrix microarrays to determine the cisplatin-induced gene expression changes in E. coli deficient in dam and mismatch repair (dam, dam mutS, and mutS mutant strains). E. coli deficient in dam are hypersensitive to cisplatin. However introducing an additional mutation in mismatch repair (i.e., a mutation in mutS or mutL) abrogates this sensitivity and essentially restores wildtype levels of resistance.
Keywords: Drug-induced expression
 
Overall design Overnight cultures were diluted 1000-fold and grown in Luria-Bertani (LB) medium until the cells reached exponential growth as determined by OD600. The exponentially growing cells were resuspended in M9 minimal medium at a cell density of 2 X 108 cells/ml in a volume of 15 ml and treated with 150 uM cisplatin for 2 hours at 37°C. Following treatment cultures were resuspended in 15 ml LB and allowed to recover for 90 minutes at 37°C. OD600 readings were taken after the recovery period, when RNA isolation began. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) according to the manufacture’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 (20mM 3-[N-morpholino]propanesulfonic acid, 5mM sodium acetate, 1mM 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.
 
Contributor(s) Robbins J, Zdraveski Z, Marinus M, Essigmann J
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Submission date Jul 25, 2005
Last update date Mar 16, 2012
Contact name Jennifer Robbins
E-mail(s) jrobbins@mit.edu
Organization name Massachusetts Institute of Technology
Department Biological Engineering
Lab John Essigmann
Street address 77 Massachusetts Ave.
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
 
Platforms (1)
GPL73 [Ecoli] Affymetrix E. coli Genome Array
Samples (24)
GSM65588 dam mock replicate 1
GSM65589 dam mock replicate 2
GSM65590 dam mock replicate 3
Relations
BioProject PRJNA91913

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