Scientists discover smallRNA that regulates bacterial infection georgiatech nature
carbenicillin. The plates were then incubated in the anaerobic chamber at 37 °C for 20 h before imaging. β-galactosidase activities were estimated as described previously. Briefly, images of colony biofilms were converted to 8 bit in ImageJ. Thresholding was carried out to define colony objects for measurements. Mean pixel intensity was measured for each colony and subtracted with the mean pixel intensity of a colony of WT PA14.
0.02 with fresh medium. Next, 10 μl diluted cultures were spotted onto 0.2-μm Nuclepore track-etched polycarbonate membranes that were placed on BHI agar plates . The plates were then incubated in the anaerobic chamber at 37 °C for 16 h. Membranes containingbiofilms were transferred to the BeadBug prefilled tubes that contained 1 ml phosphate-buffered saline . Biofilms were mechanically disrupted by bead beating for 45 s.
For static growth condition, three biological replicates for each strain were collected. Cell pellets were resuspended in lysis buffer glycerol, 1 mM Trisphosphine) , pH 7.5) with cOmplete Mini protease inhibitor cocktail . Following three rounds of sonication on ice, supernatants were clarified by sedimentation . Aliquots of each sample were reduced with TCEP, alkylated with iodoacetamide and labelled with tandem mass tags .
Liquid chromatography–tandem mass spectrometry experiments were carried out using a Dionex Ultimate 3000 rapid separation LC nano-ultraperformance LC system and a Lumos Orbitrap mass spectrometer . Separation of peptides was carried out by C18 reversed-phase chromatography at a flow rate of 300 nl minusing a Thermo Scientific reversed-phase nano EASY-spray column . Solvent A was water/0.1% formic acid, and solvent B was 80% acetonitrile/20% water/0.1% formic acid.
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