|
| Status |
Public on Aug 07, 2012 |
| Title |
T3 during hacA MT triplicate 2/3 |
| Sample type |
RNA |
| |
|
| Source name |
Mycelial biomass, exponential growth, glucose-limited bioreactor batch cultures of hacA mt
|
| Organism |
Aspergillus niger |
| Characteristics |
strain: MA70.15 (DkusA::amdS+ in AB4.1 pyrG-)
genotype/variation: hacA mutant
phase: T3 exponential growth
|
| Biomaterial provider |
Arthur Ram: Molecular Microbiology and Biotechnology group at the Institute Biology at Universiteit Leiden
|
| Treatment protocol |
Mid-exponential growth phase from bioreactor batch culture with carbon-limited (glucose) minimal medium
|
| Growth protocol |
Conidia for inoculation of bioreactor cultures were harvested from solidified CM with a sterile detergent solution containing 0.05% (w/v) Tween80 and 0.9% (w/v) NaCl. Batch cultivation of HacAWT and HacACA was initiated by inoculating 5L MM with conidial suspension to give 109 conidia L-1. Glucose was sterilized separately and added to sterile MM to give a final concentration of 0.75% (w/v). During cultivation at 30 °C, pH 3 was maintained by computer-controlled addition of 2 M NaOH or 1 M HCl. Sterile air was supplied at 1 L min-1 through a ring-sparger. Dissolved oxygen tension was above 40% of air saturation at any time, ensuring sufficient oxygen for growth. After spore germination 0.01% (v/v) polypropyleneglycol P2000 was added as antifoam agent. Submerged cultivation was performed with 6.6 L BioFlo3000 bioreactors (New Brunswick Scientific, NJ, USA).
|
| 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
|
| Description |
strain nc2.1: homologous recombination of the wild-type hacA locus in strain MA70.15 (DkusA::amdS+ in AB4.1 pyrG-) with a construct carying the constitutively active hacA locus and the AopyrG gene as selection marker
|
| Data processing |
The RMA Bioconductor package was used for data processing
|
| |
|
| Submission date |
Jul 03, 2012 |
| Last update date |
Aug 07, 2012 |
| Contact name |
Benjamin M. Nitsche |
| E-mail(s) |
bmnitsche@gmail.com
|
| Organization name |
Leiden University
|
| Department |
Institute of Biology
|
| Lab |
Molecular Microbiology and Biotechnology
|
| Street address |
Sylviusweg 72
|
| City |
Leiden |
| State/province |
The Netherlands |
| ZIP/Postal code |
2333 BE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6758 |
| Series (1) |
| GSE39070 |
Genome-wide expression analysis upon constitutive activation of the HacA bZIP transcription factor in Aspergillus niger |
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