phenotype: improved rifamycin B producer time: 36h
Growth protocol
For shake-flask experiments, vegetative mycelium was used to inoculate 500 ml baffled Erlenmeyer flask containing 50 ml of the modified RFB 2244 liquid media (50 g defatted soybean meal, 50 g D(+)-glucose monohydrate, 10 g propylene glycol, 5 g (NH4)2SO4, 1 g KH2PO4, 5 g CaCO3, 1 g MgSO4 • 7H2O, 0.01 g FeSO4 • 7H2O, 0.0033 g CuSO4 • 5H2O, 0.05 g ZnSO4 • 7H2O, 0.004 MnSO4 • 5H2O, CoCl2 • 6H2O, (NH4)6Mo7O24 • 4H2O, 4 ml silicon antifoam reagent). Cultures were incubated at 28°C with shaking at 250 rpm. For bioreactor experiments, culture were pre-inoculated in Erlenmeyer flasks and grown for 120 h; then they were inoculated into bioreactors at 10% inoculum density.
Extracted molecule
total RNA
Extraction protocol
For each time point, RNA was extracted from mycelium pellets deriving from 1-ml culture samples using the GeneElute™ total RNA Purification Kit (SIGMA), recovering it in 50 µl of Elution Solution. After extraction RNAs were quantified with a NanoDrop spectrophotometer (NanoDrop Technologies) and analyzed by capillary electrophoresis on a Agilent Bioanalyzer (Agilent). The RNA samples showing an RIN (RNA Integrity Number, a quality parameter calculated by the instrument software) value higher than 7 were processed for microarray hybridization,
Label
biotin
Label protocol
The protocol consists in cDNA synthesis by reverse transcription (starting with 10 µg RNA), followed by cDNA fragmentation with DNase I and labeling with Terminal Deoxynucleotidyl Transferase.
Hybridization protocol
The labeled cDNAs were then hybridized for 16 h at 50°C on individual GeneChips. After hybridization, GeneChips were washed and stained with streptavidin-conjugated phycoerythrin by using the Fluidic Station FS450 (Affymetrix) following the FS450_0005 Protocol. Fluorescent images of the microarrays were acquired using a GeneChip Scanner 3000 (Affymetrix).
Scan protocol
Chips were scanned through GeneArray Scanner 3000 7G (Affymetrix) at an emission wavelenght of 560nm.
Description
improved rifamycin B producer (HP-130)
Data processing
The quality of the raw data obtained from microarray hybridization was assessed considering the MAS5.0 (Microarray Suite/Software, Affymetrix) control parameters after a global scaling at a target intensity of 100. Quality and control parameters as well as box plots of raw intensities indicated the overall high quality of the data set and the absence of any outlying sample. Probe level data was converted to expression values using the Robust Multi-array Average (RMA) procedure. Genes characterized by a statistically significant modulation of the expression level during the growth time course (within-class temporal differential expression) were identified using the EDGE software package, which is based on the Optimal Discovery Procedure and allows identifying genes that are differentially expressed between two or more different biological conditions or to perform significance analysis on time course experiments
