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, 1995). In the resulting P.?aeruginosa strain PAO6738, the PA4129-PA4134 deletion was confirmed by PCR, and the PCR fragment obtained was checked by sequencing. P.?aeruginosa strain PAO6739 (��PA4129-PA4134, ��cioAB) was constructed analogously in a PAO6437 (��cioAB) background. A translational PA4130'-��lacZ fusion was constructed by inserting a 593?bp BamHI-PstI fragment carrying the proximal part of PA4130 into the BamHI-PstI sites of pME6013. This fragment was generated by PCR amplification using PAO1 genomic DNA and primers P4130FW and P4130RV. HCN was quantified in P.?aeruginosa culture supernatants as previously described (Gewitz et?al., 1976). NADH-dependent oxygen uptake was assayed in whole cells using a Clark-type oxygen electrode as described previously (Cunningham and Williams, 1995). ��-galactosidase specific activities were determined by the Miller method (Miller, 1972). Pseudomonas aeruginosa Azastene PAO1 and PAO6344 were inoculated at an OD600 of 0.01 into 30?ml of MMC in hermetically closed 125?ml bottles. The cultures where grown at 37��C with gentle shaking (130?r.p.m.) until they reached an OD600 of approximately 1.0; cells were harvested and RNA protect bacteria (Qiagen, Milan, Italy) was added. Total RNA was isolated by the hot phenol method as described elsewhere (Leoni et?al., 1996), followed by DNaseI treatment (Roche, Milan, Italy). The quality of total RNA was investigated by agarose gel electrophoresis and an RNA 6000 Nano LabChip in an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Next, cDNA VE-821 clinical trial synthesis was obtained with random primers and Superscript II reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA) using 10?��g of total RNA. cDNA fragmentation, labelling, hybridization, staining and washing steps were performed according to the manufacturer's protocol for the Affymetrix P.?aeruginosa GeneChip arrays (Affymetrix, Inc., Santa Clara, CA, USA). The arrays were scanned with the Affymetrix GeneChip Scanner 3000. Processing of the P.?aeruginosa GeneChip (Affimetrix) was performed at the University of Lausanne, Center click here for Integrative Genomics. For each condition tested, cultures were grown in triplicate, and RNA from these cultures was pooled before proceeding to cDNA synthesis. In addition, biological replicates for each condition were performed on a separate day and run on a different microarray chip. Data were analysed as previously described (Gaillard et?al., 2008). Induced and repressed genes (Table?1) meet the following criteria: (i) the P-value obtained for each transcript analysed was