Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of Actinosynnema mirum grown in GYM medium compared to growth in TB medium.

ABSTRACT: Actinosynnema mirum cells were grown in 3 biological replicates and harvested in exponential growth phase either grown in terrific broth (TB) or in glucose-yeast-malt (GYM) medium to analyse the differential gene expression under different grwoth media. A preculture of A. mirum was grown in 20 mL NL148 medium in 100 mL shake flasks for 3 days at 28 °C and 180 rpm (2.5 cm shaking diameter). 1 mL of the preculture was transferred to 60 mL terrific broth (TB) or 60 mL glucose-yeast-malt (GYM) medium in 500 mL shake flasks cultivated for 20 h at 28 °C and 180 rpm (2.5 cm shaking diameter). Pre and main culture were cultivated in triplicates. 495 µL of cultures was transferred to 96 well plates and 5 µL ritonavir in a final concentration of 0.1 mg mL-1 was added. Samples were incubated at 28 °C, 200 rpm for 44 h. Samples were centrifuged at maximum speed for 30 s, the supernatant was removed, and samples were flash frozen in liquid nitrogen. Cells were mechanically homogenized in 1X DNA/RNA Shield™ using Bead Tubes Type B (Macherey-Nagel, Düren, Germany) in a Bead Tube Holder at maximum speed for 20 min. RNA isolation was performed using the Quick-RNA Miniprep Plus (ZYMO research, Freiburg, Germany). An additional in-column DNase I treatment was performed. RNA pellets were eluted in 100 µL DNase/RNase-free water. The purified RNA was quantified with a NanoDrop (Thermo Scientific™, Waltham, USA). RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was stored at -80 °C. Total-RNA with an RNA integrity number (RIN) > 9 was used to prepare cDNA libraries. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. Preparation of cDNA libraries were performed according to the manufacturer’s instructions of TruSeq stranded mRNA Kit (Illumina, San Diego, USA). Subsequently, each cDNA library was sequenced on a HiSeq1500 (2 x 75nt PE rapid v2) Sequencer system (Illumina, San Diego, USA).

INSTRUMENT(S): Illumina HiSeq 1500

ORGANISM(S): Actinosynnema mirum DSM 43827

SUBMITTER: Lisa-Marie Schmitz

PROVIDER: E-MTAB-10100 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To elucidate the effect of the rational ribosomal engineering on the changes in the expression of the endogenious biosynthetic gene clusters the transcriptome analysis was performed. The Streptomyces albus strains carrying mutations in rpsL gene (encode for ribosomal protein S12) and the deletion of the rsmG gene (16S rRNA methyltransferase G), as well as their combination were used for the experiment. The list of the strains with mutations is next: S. albus K88E, S. albus GI92, S. albus K88E-GI92, S. albus P91S, S. albus K88E-P91S, S. albus del rsmG, S. albus K88E-GI92 del rsmG, S. albus K88E-P91S del rsmG. Abovementioned strains along with S. albus native strain were grown in NL-19 production medium. Samples were harvested by centrifugation after 48 and 72 hours of cultivation. For total RNA isolation, S. albus cells were grown in SG medium (for ara expression) or NL19 medium (for indigenous BGC expression). Then, 2 ml of 2-day and 3-day cultures was spun down for 20 s at 14,000 × rpm, and the pellets were immediately frozen in liquid nitrogen and stored at ?80 °C. Total RNA extraction was performed using an RNeasy Kit (Qiagen, Hilden, Germany) as previously described (Huser et al. 2003). An RNase-Free DNase set (Qiagen) was used two times for on-column DNA digestion, and an additional DNase treatment was then performed with a DNase I kit (Roche Diagnostics, Mannheim, Germany) to ensure that all DNA was completely removed. To check the RNA samples for DNA contamination, PCR was performed using oligonucleotides designed to create two different products approximately 150 bp and 500 bp in size. Initially, RNA quality was checked with Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A Ribo-Zero rRNA Removal Kit for bacteria was obtained from Illumina (San Diego, CA, USA) and used to remove ribosomal RNA molecules from isolated total RNA. rRNA removal was checked using an Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries (Koepff et al. 2017). The resulting cDNAs were pair-end sequenced on an Illumina HiSeq 1500 system (San Diego, CA, USA) using a 70 bp read length. Short read alignments and differential gene expression were illustrated using ReadXplorer 2.2.0 (Hilker et al. 2014) and DEseq (Anders and Huber 2010), respectively.

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RNA-seq of Actinosynnema mirum grown in GYM medium compared to growth in TB medium.

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