Comment[ArrayExpressAccession]	E-MEXP-3032
Investigation Title	Response of Streptomyces coelicolor to treatment with the antibiotics vancomycin bacitracin and moenomycin A
Comment[Submitted Name]	Response of Streptomyces coelicolor to treatment with the antibiotics vancomycin bacitracin and moenomycin A
Comment[AEExperimentDisplayName]	Response of Streptomyces coelicolor to treatment with the antibiotics vancomycin bacitracin and moenomycin A
Comment[MIAMExpressLogin]	ahesketh
Comment[MIAMExpressSubmissionID]	7175
Experiment Description	To determine the response at the transcriptional level of S. coelicolor cells to treatment with 3 antibiotics that target distinct stages of cell wall biosynthesis, biological triplicate cultures of strain M600 were treated with sub-lethal concentrations (10 µg/ml) of vancomycin, bacitracin or moenomycin A. Samples were taken 0, 30, 60, 90 minutes after addition of the antibiotic. A negative control which received no drug treatment was also performed.
Experimental Design	in_vivo_design	co-expression_design	time_series_design	stimulus_or_stress_design
Comment[AEExperimentType]	transcription profiling by array
Experimental Factor Name	COMPOUND	DOSE	TIME
Experimental Factor Type	compound	dose	time
Person Last Name	Hesketh
Person First Name	Andrew
Person Mid Initials	R
Person Email	arh69@cam.ac.uk
Person Phone	 +44 1223 760243
Person Affiliation	Univeristy of Cambridge, Biochemistry
Person Address	80 Tennis Court Road, Cambridgeshire CB2 1GA
Person Roles	submitter
Person Roles Term Source REF	MGED Ontology
Quality Control Type	biological_replicate
Public Release Date	2011-04-29
Comment[ArrayExpressSubmissionDate]	2010-12-07 12:01:17
Publication Title	Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope
Publication Status	submitted
Protocol Name	P-MTAB-18155	P-MTAB-18156	P-MTAB-18157	P-MTAB-18158	P-MTAB-18159	P-MTAB-18160
Protocol Description	Purified total RNA (10 mg) was processed into labelled and fragmented cDNA for hybridisation to Streptomyces diS_div712a GeneChip arrays according to the manufacturer's published protocol for Pseudomonas aeruginosa (www.affymetrix.com).  Briefly, cDNA synthesis was performed using 72% GC-content random primers and Superscript III reverse transcriptase (Invitrogen). The resulting cDNA was fragmented to approximately a 50-200bp size range by partial digestion with DNaseI, and then terminally labelled with biotin using terminal transferase in the presence of biotinylated ddUTP. <br>(Parameters: Amount of nucleic acid labeled = 10, Label used = biotin, Amplification = PCR, Mass unit = Micro gram)	Purified total RNA (10 mg) was processed into labelled and fragmented cDNA for hybridisation to Streptomyces diS_div712a GeneChip arrays according to the manufacturer's published protocol for Pseudomonas aeruginosa (www.affymetrix.com)i.e. Affymetrix ProkGE-WS2.  <br>(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 5, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 50)	.CEL file data processed using RMA, and filtered to remove non-Streptomyces coelicolor probe sets	Biological triplicate liquid cultures were treated by adding a sub-lethal concentration (10 µg/ml) of vancomycin, bacitracin or moenomycin A and samples taken 0, 30, 60, 90 minutes after addition of the antibiotic. A negative control which received no drug treatment was also performed. 	Mycelia from liquid cultures was immediately treated with twice the volume of RNA protect bacteria solution (Qiagen) according to the manufacturer's instructions to stabilize the RNA content of the cells, and the resultant cell pellet stored at 80oC prior to processing for RNA extraction. Total RNA was isolated from mycelia harvested and stored from liquid cultures using the RNeasy midi kit from Qiagen largely according to the manufacturers' instructions, but with several modifications. Cell pellets were resuspended in TE buffer (1 ml) containing 15 mg ml-1 lysozyme and incubated for 60 min at room temperature. After addition of RNeasy RLT buffer (4 ml) samples were sonicated for 3 cycles of 20 seconds (Sanyo Soniprep 150, amplitude 18 microns), resting on ice for 1 min between bursts, then extracted twice with phenol-chloroform (4 ml) and once with chloroform (4 ml). Extracts were then treated with ethanol (2.8 ml) and applied to the RNeasy midi columns for purification according to the supplied protocol, including an on-column DNaseI digestion step for 60 min at room temperature. Purified RNA was finally eluted in 300 ml RNase-free water. <br>(Parameters: Extracted product = total_RNA, Amplification = none)	Spores of S. coelicolor M600 were germinated and grown to mid-log phase (OD600nm approx. 0.5) in NMMP as described previously (Hong et al. 2004. Characterization of an inducible vancomycin resistance system in Streptomyces coelicolor reveals a novel gene (vanK) required for drug resistance. Mol Microbiol 52: 1107-1121). <br>(Parameters: time unit = seconds, min temperature = 30, temperature unit = C, media = NMMP)
Protocol Type	labeling	hybridization	bioassay_data_transformation	specified_biomaterial_action	nucleic_acid_extraction	grow
Protocol Term Source REF	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology	MGED Ontology
Term Source Name	MGED Ontology	ArrayExpress
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MEXP-3032.sdrf.txt
PubMed ID	21569315
Publication Author List	Hesketh, A., Hill, C., Mokhtar, J., Novotna, G., Bibb, M., and Hong, H.-J.
Publication DOI	10.1186/1471-2164-12-226