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Sample GSM4446424

Query DataSets for GSM4446424

Status

Public on Mar 11, 2021

Title

Parental strain (after oxygen limitation) rep 2

Sample type

SRA

Source name

Parental strain (after oxygen limitation)

Organism

Gluconobacter oxydans

Characteristics

strain: 621H
genotype: delta-hsdR

Growth protocol

G. oxydans 621H and mutant strains were cultivated in mannitol medium containing 220 mM (4% w/v) mannitol, 5 g l-1 yeast extract, 2.5 g l-1 MgSO4 x 7 H20, 1 g l-1 (NH4)2SO4 and 1 g l-1 KH2PO4. The initial pH value of the medium was 6.0. G. oxydans possesses a natural resistance towards cefoxitin; as a precaution to prevent bacterial contaminations, cefoxitin was added to the media at a concentration of 50 μg ml-1. Precultures were grown in baffled shaking flasks at 30°C and 140 rpm. Main cultures were cultivated in 250 ml of the same medium in a bioreactor system (DASGIP, Jülich, Germany) composed of four 400-ml vessels, each equipped with electrodes for measuring the dissolved oxygen concentration (DO) and the pH. The system enabled these two parameters to be kept constant. The carbon dioxide concentration in the exhaust gas was measured continuously by an infrared spectrometer and the oxygen concentration by a zirconium dioxide sensor. The oxygen availability was kept constant at 15% DO by mixing air, O2 and N2. Calibration was performed by gassing with air (100% DO) and 100% N2 (0% DO). Anaerobic conditions were achieved by gassing with 100% N2. The agitation speed was kept constant at 900 rpm. Controlling and recording of all data as well as calculation of oxygen transfer rates (OTR) and CO2 transfer rates (CTR) was carried out by the “Fedbatch Pro” software (DASGIP, Jülich, Germany). For oxygen limitation experiments, the bacteria were first cultivated at 15% DO to an optical density at 600 nm (OD600) of 2.5 and then the culture was supplied with a gas mixture composed of 2% O2/98% N2

Extracted molecule

total RNA

Extraction protocol

RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany) and quality-checked using the Agilent Tape station 2200 (Agilent Technologies, Waldbronn, Germany) with the RNA ScreenTape according to the manufacturer´s instructions.
To remove rRNA of all samples, the RiboMinus Transcriptome Isolation Kit for Yeast and Bacteria (Invitrogen, Carlsbad, USA) was used. rRNA-depleted RNA was precipitated with ethanol overnight at -20°C. The pellet was dissolved in 7 µl nuclease-free water. Successful depletion was verified with the Tape Station using Agilent´s High Sensitivity RNA ScreenTape. Sequencing libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Frankfurt, Germany) following the protocol for rRNA-depleted RNA using 11 cycles for PCR amplification of adaptor-ligated DNA. For quantification of the final cDNA libraries, the KAPA library quantification Kit from KapaBiosystems (Roche, Unterhaching, Germany) was used following the manufacturer´s instructions. The qPCR itself was conducted on a qTOWER 2.2 (Analytik Jena, Jena, Germany).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Read processing and strand-specific mapping to the G. oxydans 621H reference genome (NC_006672.1-NC_006677.1) was carried out with the CLC Genomics Workbench (Qiagen, Aarhus, Denmark).
Empirical analysis of differential gene expression for three biological replicates was performed with EdgeR
Genome_build: NC_006672.1-NC_006677.1
Supplementary_files_format_and_content: Results of differential gene expression analysis with edgeR as tab limited text file.
Supplementary_files_format_and_content: EdgeR result matrix with fold change values, p-values and absolute measurements for every gene per sample

Submission date

Mar 31, 2020

Last update date

Mar 11, 2021

Contact name

Angela Kranz

E-mail(s)

a.kranz@fz-juelich.de

Organization name

Forschungszentrum Jülich GmbH

Department

IBG-1: Biotechnology