Arthur Ram: Molecular Microbiology and Biotechnology group at the Institute Biology at Universiteit Leiden
Treatment protocol
Day 1 post-carbon starvation during maltose-limited submerged batch cultivation
Growth protocol
Conidial suspensions for inoculation of bioreactor cultures were prepared by growing the A. niger laboratory strain N402 (cspA1 derivative of ATCC9029) (Bos et al., 1988) on solidified (1.5% agar) complete medium (CM) for three days at 30◦C in the dark. Spores were harvested with sterile physiological salt solution (0.9% NaCl) and filtered through Myracloth (Calbiochem, San Diego, CA, USA) to retain mycelial debris and solidified medium. CM contained per liter: 10 g glucose, 6 g NaNO3 , 1.5 g KH2PO4 , 0.5 g KCl, 0.5 g MgSO4 · 7H2O, 1 g casamino acids, 5 g yeast extract and 1 ml trace metal solution. e pH was adjusted to 5.8 with NaOH. e trace metal solution, modi ed from Vishniac et al. (1957), contained per liter: 10 g EDTA, 4.4 g ZnSO4 · 7H2O, 1.01 g MnCl2 · 4H2O, 0.32 g CoCl2 · 6H2O, 0.315 g CuSO4 · 5H2O, 0.22 g (NH4) · 6Mo7O24 · 4H2O, 1.47 g CaCl2 · 2H2O and 1 g FeSO4 · 7H2O. Minimal medium (MM) for bioreactor cultivations contained per liter: 4.5 g NH4Cl, 1.5 g KH2PO4 , 0.5 g KCl, 0.5 g MgSO4 · 7H2O and 1 ml trace metal solution. e pH was set to 3 with HCl. Aer autoclavation, 16 ml of heat-sterilized 50% (w/v) maltose monohydrate solution were added per kg of MM. Batch cultures were performed in 6.6 L BioFlo3000 bioreactors (New Brunswick Scientific) as previously described by Jørgensen et al. (2010). Briefly, autoclaved bioreactor vessels were filled with 5 L (kg) sterile MM. During cultivation at 30◦C, the controller was set to maintain pH 3 by addition of titrants (2 M NaOH and 1 M HCl). Sterile air was supplied at a rate of 1 L per min. Prior to inoculation, 1.5 ml of 10% (w/v) filter-sterilized yeast extract was added to enhance conidial germination. Cultures were inoculated with freshly harvested spore suspensions to give 10 to the power of 9 conidia per liter. To reduce the loss of hydrophobic conidia during germination, the stirrer speed was set to 250 rpm and the culture was aerated via the headspace during the first six hours aer inoculation. Subsequently, the stirrer speed was increased to 750 rpm, 0.5 ml of polypropyleneglycol P2000 was added as antifoam agent and air was supplied via the sparger.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from homogenized mycelial samples using TRIzol reagent (Invitrogen).
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
Data processing
The RMA Bioconductor package was used for data processing
