Computational protocol: Novel Two-Component Systems Implied in Antibiotic Production in Streptomyces coelicolor

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Protocol publication

[…] For RNA extraction from S. coelicolor wild-type and ΔabrC1/C2/C3 mutant strains, NA plates covered with a cellophane sheet were inoculated with 7.5×106 spores and incubated at 30°C for 50 h. Prior to RNA isolation using a RNeasy Midi Kit (Quiagen) the mycelia was harvested and suspended in RNA-protect Bacteria Reagent (Qiagen). An additional step with RNase free DNase (Qiagen) was incorporated to remove any contaminating DNA. The quality and concentration of RNA were assayed using gel electrophoresis and spectrophotometer assays (Q-bit and Agilent bioanalizer). Four biological replicates were used.cDNA versus gDNA microarrays experiments were chosen due to the advantages described elsewhere , . The S. coelicolor SCo40 microarrays used were obtained from the Functional Genomics Laboratory of Surrey University (UK) . The Pronto! Universal Microarray Hybridization kit (Corning, # 40026) was used for pretreatment and prehybridization. Cy3-cDNA and Cy5-gDNA labelling reactions were performed according to the recommendations described by . Hybridization assays were done as in Rodríguez-García et al. and TIFF images were generated by Genepix DNA Microarray Scanner 4000B and processed with Genepix Pro 4.0 software. Bioconductor software package limma (linear models for microarray analysis) and rank products were used to analyse and assess the statistical significance of the data , . Background correction was applied using the normexp function. Then, the log of Cy3/Cy5 intensities were normalized using block-weighted medians and global loess. The different p-values of the contrast between both strains were corrected for multiple testing FDR (false discovery rate) or by the rank products pfp method (proportion of false positives). To consider a gene differentially expressed, it should have passed at least one of these criteria: limma FDR-corrected p-value<0.05 or rank products pfp value<0.05. All data is MIAME compliant and the raw data has been deposited in a MIAME compliant database (ArrayExpress, accession number E-MEXP-2841) […]

Pipeline specifications

Software tools GenePix Pro, limma
Application Gene expression microarray analysis
Organisms Streptomyces coelicolor, Streptomyces coelicolor A3(2)
Chemicals Calcium