strain background: N402 genotype/variation: TupA mutant (UV-mutant RD15.8#36) time point: whole fungal mycelium time point: exponential growth
Growth protocol
Bioreactor cultivations were carried out as described previously. Fermentation medium (FM) adjusted to pH 3, is composed of 0.75 % glucose, 0.45 % NH4Cl, 0.15 % KH2PO4, 0.05% KCl, 0.05 % MgSO4, 0.1 % trace element solution and 0.003 % yeast extract as described [41]. Freshly harvested conidia (5 x 109) from strain N402 and RD15.8#36 were used to inoculate 5 liters of FM. Cultivations were performed in a BioFlo3000 bioreactor (New Brunswick Scientific), where the temperature, pH (set to 3), and agitation speed were controlled online using the program NBS Biocommand. The cultivation program consisted of two consecutive phases: (i) 30°C, agitation speed of 250 rpm, and headspace aeration, for the first 5 h; (ii) 30°C, agitation speed of 750 rpm, and sparger aeration during the second phase. Mycelial samples were taken after specific time points for microarray, metabolic, and microscopic analyses.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from homogenized mycelial samples using TRIzol reagent (Invitrogen) followed column purification including a DNAse I treatment.
Label
biotin
Label protocol
The Affymetrix One-Cycle Target Labeling Kit and Control Reagents (#900493) are used to synthesize Biotin-labeled cRNA. From each RNA sample 2 μg is used for the labeling experiments.
Hybridization protocol
The GeneChip Hybridization, Wash and Stain Kit (#900720) is used for the for the hybridizations, washing, staining and scanning of the chips. The Affymetrixprotocols are strictly followed. The Affymetrix custom Aspergilus niger Genome Arrays are used for hybridization. To the 270 μl hybridization cocktail, 30 μl labeled material is added according the manual of the Affymetrix One Cycle Target Labeling Kit.
Scan protocol
Standard Affymetrix protocol
Description
101281-01.CEL
Data processing
RMA background correction, normalization and probe summarization steps were performed according to the default setting of the RMA/affy package (v.1.38.1) as implemented in Bioconductor.
The transcriptional repressor TupA in Aspergillus niger is involved in controlling gene expression related to cell wall biosynthesis, development, and nitrogen source availability
