S. coelicolor A3(2) strain M145 was maintained on SMMS agar (Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK) for spores. For liquid culture, spores were pre-germinated in 2xYT medium (Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK) and inoculated into SMM liquid medium (Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK) at a density of ~10exp7 spores/ml. Baffled 2L flasks containing stainless steel coils with 350 ml working volume were used for liquid cultures. Cultures were incubated at 30°C in an orbital shaker at 300 rpm. For measuring cell growth, samples were collected at regular intervals and mixed with one-eighth volume of 2.5 M HCl and then subjected to sonication to break mycelial pellets (Brana, A. F., S. Wolfe, and A. L. Demain. 1986. Relationship between nitrogen assimilation and cephalosporin synthesis in Streptomyces clavuligerus. Arch Microbiol 146:46-51). Absorbance of the resulting solution at 450 nm was then taken as a measure of growth. Proper dilution was made to ensure an OD450 measurement below 1, which is in the linear range of detection.
Extracted molecule
total RNA
Extraction protocol
Immediately after culture samples were collected, one-fifth volume of an ice-cold stop solution (5% phenol in ethanol) (Bernstein, J. A., A. B. Khodursky, P. H. Lin, S. Lin-Chao, and S. N. Cohen. 2002. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc Natl Acad Sci U S A 99:9697-702) was added to the sample volume to prevent RNA degradation. The sample was quickly centrifuged at 4°C and the supernatant was discarded. The remaining sample was then stored at -80°C. To isolate RNA, the frozen pellets were transferred to a mortar and pestle and ground to fine powder in the presence of liquid nitrogen. RLT solution from RNeasy Mini Kit (Qiagen Inc., Valencia, CA) was added before the ground pellets thawed. The remaining steps for RNA isolation were carried out as per manufacturer’s instructions. RNA integrity was determined by gel electrophoresis while its quantity and purity were estimated by UV absorbance at OD260 and OD260/OD280, respectively. Total RNA was reverse transcribed to cDNA by SuperscriptII (Invitrogen, Carlsbad, CA) with random hexamers (IDT, Coralville, IA) incorporating aminoallyl-dUTP (Ambion, Austin, TX).
Label
A647
Label protocol
The cDNA was then incubated with Alexa 647 (Invitrogen, Carlsbad, CA) for labeling as per manufacturer's instructions.
Spores for S. coelicolor M145 were generated on Mannitol-Soy flour or R5 agar Cultures for genomic DNA preparation were performed in YEME medium with 0.5% glycine supplement at 30°C until early stationary phase (refer to T Kieser, MJ Bibb, MJ Buttner, KF Chater, DA Hopwood: Practical Streptomyces Genetics: The John Innes Foundation, Norwich, UK; 2000).
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA (gDNA) extraction was carried out using Kirby mix procedure as described elsewhere (T Kieser, MJ Bibb, MJ Buttner, KF Chater, DA Hopwood: Practical Streptomyces Genetics: The John Innes Foundation, Norwich, UK; 2000). About 500 µl of 20 µg/µl gDNA was sonicated briefly for 30-40 sec for shearing them to ~500 bp average size (confirmed by gel electrophoresis).
Label
Cy3
Label protocol
The DNA was labeled with Label IT® Cy3 Labeling Kit (Mirus Bio Corp., Madison, WI) according to suppliers instructions.
Hybridization protocol
The labeled cDNA and gDNA were then mixed and co-hybridized to microarray slides in the presence of 50% formamide. After 16 hours incubation at 50°C, slides were washed and scanned.
Scan protocol
Arrays were scanned using ScanArray5000 (Perkin Elmer, Wellesley, MA). Images were analyzed using ImaGene (BioDiscovery, El Segundo, CA) to obtain raw intensity data for each spot. The median fluorescence intensity from each spot was used for all subsequent analysis.
Data processing
A quantile normalization method was employed to normalize these log2 ratios from each hybridization (S Mehra, W Lian, KP Jayapal, SP Charaniya, DH Sherman, WS Hu: A framework to analyze multiple time series data: a case study with Streptomyces coelicolor. J Ind Microbiol Biotechnol 2006, 33:159-72). The resulting log2 ratios were not only used to compare transcript levels of the same mRNA species in different biological samples, but also used as an estimate of the relative abundance of each mRNA species.
Submission date
Jun 16, 2010
Last update date
Oct 25, 2010
Contact name
Marlene Castro-Melchor
Organization name
University of Minnesota
Department
Department of Chemical Engineering and Materials Science
