Axenic cultures of the cyanobacterium Synechocystis sp. PCC 6803 were grown photoautotrophically under continuous illumination of 175 µmol photons/m2 s (warm white fluorescent tubes, Osram L 32) at 29° C. For isoprene production, cultures were pre-cultivated at high CO2 (5%) in BG11 medium. After 24 h, the pre-cultures were used to inoculate the main cultures in closed 0,1 L Schott flasks at OD750 of about 1 in 50 ml of BG11 supplemented with 50 mM NaHCO3. The cultures were incubated at 30° C, with an illumination of about 150 µmol photons/m2 s under continuous stirring with 150 rpm. After 24 h, samples were taken for RNA isolation
Extracted molecule
total RNA
Extraction protocol
Synechocystis 6803 cells were collected by centrifugation (4000 rpm, 4°C, 4 min) and the cells were dissolved in 500 µl PGTX solution (Pinto et al., 2009) with the following composition: 39.6% (w/v) phenol, 7% (v/v) glycerol, 7 mM 8-hydroxyquinoline, 20 mM EDTA, 97.5 mM sodium acetate, 0.8 M guanidine thiocyanate, 0.48 M guanidine hydrochloride. The samples were incubated for 15 min at 65°C and put on ice for 5 minutes. After addition of 500 µl chloroform/isoamylalcohol (24:1), samples were incubated at room temperature for 10 minutes. Samples were centrifuged at 6,000 rpm, 20°C for 10 min. The upper aqueous phase was transferred into a new tube and the same volume of chloroform/isoamylalcohol (24:1) was added and mixed. Samples were centrifuged as described above and the aqueous phase removed again and combined with an equal volume of isopropanol. After gently inverting the tube, RNA was precipitated over night at -20°C. RNA was pelleted through centrifugation (13,000 rpm, 4°C, 30 min). The pellet was washed with 1 ml of 70% ethanol (13,000 rpm, 20°C, 5 min), allowed to air dry for approximately 10 min and resuspended in 30 µl H2O.
Label
Cy3
Label protocol
The RNA was labeled directly, without cDNA synthesis with the Kreatech “ULS labeling kit for Agilent gene expression arrays” with Cy3 according to the manufacturers protocol.
Hybridization protocol
The labelled RNA was fragmented and hybridized as described by the manufacturer's instructions for Agilent one color microarrays
Scan protocol
Arrays were scanned on the Agilent Technologies Scanner G2505C US90900275, using Agilent Feature Extraction Software 10.7.3.1 and the protocol GE1_107_Sep09 for one-color arrays
Data processing
Raw data were processed with the R package Limma. Median signal intensity was normexp background corrected and quantile normalized