![]() After purification and DNAse treatment, samples were ready for RNA sequencing. Possible remaining DNA was removed via DNA-free™ DNA Removal Kit (Invitrogen) according to the manufacturer's instructions. Samples were purified using the innuPREP RNA Mini Kit 2.0 (Analytik Jena) according to the manufacturer's instructions. RNA was isolated with the Trizol reagent (Invitrogen) using frozen ground mycelium according to the manufacturer's instructions. This filter was wrapped in tinfoil and immediately frozen in liquid nitrogen. ![]() The biomass sample was gained by filtrating the culture broth sample via vacuum filtration, collecting mycelium on a filter. Cultivation was continued as a batch cultivation. In steady state, the carbon source was changed to cellulose (autoclaved separately) by removing 500 g culture broth and subsequently adding 450 g bioreactor medium (without glucose) and 50 g microcrystalline cellulose resulting in 5 kg total weight of the culture broth. To prevent foaming, PPG 2000 was added with a rate of 20 mg/h during chemostat cultivation. At the end of the exponential phase when glucose concentration was close to 0-1 g/L (~13 g base addition) chemostat cultivation was started by addition of medium at a dilution rate of 0.1 1/h and maintaining culture broth weight at 5 kg. Aeration and stirring were constantly increased within 10 h to 1 slpm and 750 rpm, respectively. After inoculation, a time-based profile for aeration and stirring was started. pH regulation was achieved via 25 % ammonia solution and 20 % phosphoric acid. Prior to inoculation, the temperature was set to 45 ☌, pH value to 6.7 (if necessary), stirring to 100 rpm, and aeration to 0.01 slpm (after reaching 100 % DOT). Prior to autoclaving pH was set to 6.7 using 10 M NaOH. Biotin was sterile filtrated and added to the medium after autoclaving. CaCl2x2H2O, glucose and trace elements were autoclaved separately and added to the medium after autoclaving. Samples for RNA extraction were taken in exponential state (ex), steady state (SS) and after (t1=0.5 h, t2=1 h, t3=2 h, t4=4 h) the addition of the medium containing cellulose.ġ*10^9 spores/L were inoculated in bioreactor medium containing 76 mM (NH4)2SO4, 2 mM MgSO4x7H2O, 12 mM KH2PO4, 7.5 mM KCl, 0.27 mM CaCl2x2H2O, 0.025 mM biotin, 55.5 mM glucose and trace elements (134 µM EDTA disodium salt dihydrate, 70 µM ZnSO4x7H2O, 162 µM H3BO3, 23 µM MnSO4xH2O, 16.4 µM FeSO4x7H2O, 6.5 µM CoCl2圆H2O, 5.8 µM CuSO4x5H2O, 5.7 µM Na2MoO4x2H2O pH adjusted to 6 using 1 M NaOH). GEO help: Mouse over screen elements for information.Ĭondition: t3= 2 h after spiking cellulose in steady state
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