Illumina MiSeq paired end sequencing; RNA-Seq of Corynebacterium glutamicum ATCC13032 differing in growth rates reveals the growth rate modulon
RNA-Seq of Corynebacterium glutamicum ATCC13032 differing in growth rates reveals the growth rate modulon
Samples of 2 mL were withdrawn from the bioreactor and immediately centrifuged at 20000 g for 30 seconds at 4 °C. The supernatant was discarded and the remaining cell pellet was immediately flash frozen in liquid nitrogen. C. glutamicum ATCC13032 glycerol stocks were plated on 2 x Tryptone yeast extract (2xTY) agar plates. After 60 h of incubation, glass reaction tubes containing 5mL of 2xTY medium were inoculated with colonies harvested from the plates. After 8 h on a rotary shaker at 120 RPM, these cells then were used to inoculate shaking flasks containing modified CGXII minimal medium and glucose. The minimal medium was modified so that either phosphate, nitrogen or carbon was the limiting substrate. These cultures were kept 84 h (Phosphate), 60 h (Carbon), 48h (Nitrogen) on a rotary shaker at 120 RPM and then were used to inoculate fresh CGXII medium containing 5 % Glucose and 30 mg/L protocatechuic acid in a 1.5 L bioreactor. In the bioreactor growth conditions were kept constant at 30 ° C, pH = 7.4 and dissolved oxygen levels above 30 % of the physical maximum and the growth rate transitions were sampled. Growth limitation pre-cultures prior to bioreactor cultivation: For the carbon limitation the content of six such glass reaction tubes was used to inoculate six baffled 500 mL shaking flask containing 50 mL modified CGXII minimal medium (Buchholz et al. 2014) each supplemented with 1 % (w/v) glucose. The carbon limited pre culture was harvested 60 hours after inoculation. For the phosphate limitation the content of one glass reaction tube was used to inoculate a 3 L shaking flask with 300 mL of modified CGXII minimal medium in which the concentration of K2HPO4 and KH2PO4 was reduced to 0.01 g L 1. The phosphate limited pre culture was harvested 84h after inoculation. For the nitrogen limitation the content of one glass reaction tube was used to inoculate 300 mL of modified CGXII minimal medium without urea. The concentration of MOPS buffer was doubled to 42 g L 1 and the concentration of NH4SO4 was reduced to 0.1 g L 1. The nitrogen limited pre culture was harvested 48 hours after inoculation. Harvested samples were then used to inoculate fresh CGXII medium containing 5 % Glucose and 30 mg/L protocatechuic acid in a 1.5 L bioreactor.  Total RNA was isolated from three biological replicates using RNeasy Mini Kit along with a DNase Kit (both from Qiagen).  Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. 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. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries.
Experimental Factor: stimulus
phosphate limitation
Experimental Factor: growth condition
growth rate 0.4 _diff [1/h]
Experimental Factor: time
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