title: NextSeq 500 paired end sequencing; Various evolutionary trajectories lead to loss of activity of tobramycin-potentiating compounds in Burkholderia cenocepacia biofilms
design: Various evolutionary trajectories lead to loss of activity of tobramycin-potentiating compounds in Burkholderia cenocepacia biofilms
library_construction_protocol: Start population: this sample was collected from an overnight culture. Evolved populations: after 15t cycles of biofilm formation - treatment with TOB alone, TOB+BH, TOB+ECO or TOB+MICO - planktonic regrowth (48h), cells were allowed to regrow for 48 hours. These cells were used to extract DNA from. Three independent lineages of biofilms were set up, to avoid contamination. Biofilms were formed on beads, starting from a planktonic culture with approximately 10^6 cells. Biofilms were allowed to grow for 24 hours After 24 hours, biofilms were treated with either tobramycin (TOB) (768ug/ml) alone or in combination with baicalin hydrate (BH) (250uM), econazole (ECO) (1uM) or miconazole (MICO) (1uM). An untreated control was also included. After 24 hours of treatment, the remaining cells were divided in two, where one part was used to quantify the remaining cells and the other part was allowed to regrow planktonically. This process was repeated 15 times. DNA  was extracted using a modified bead-beater protocol. Briefly, the pellets were resuspended in 200 µl TE-buffer (10 mM Tris-HCl (Roche Diagnostics, Mannheim, Germany), 1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), pH 8.0). 100 µl of this suspension was transferred to a 2 ml Eppendorf tube containing approximately 0.5 ml of 0.1-mm-diameter glass beads (Sigma-Aldrich), 0.5 mg/ml pronase (Roche) and 500 µl lysis buffer (50 mM Tris-HCl, 70 mM EDTA, 1% sodium dodecyl sulfate (SDS; Sigma-Aldrich), pH 8). After 10 sec of vigorous vortexing, the samples were incubated for 1 hour at 37°C. Afterwards, the tubes were briefly centrifuged and 200 µl saturated ammonium acetate was added to the lysate. The tubes were, again, vigorously vortexed for 10 sec and centrifuged (13000 rpm, 2 min). Subsequently, 600 µl chloroform (Sigma-Aldrich) was added and the tubes were mixed vigorously. Proteins and polysaccharides were removed by centrifugation (13000 rpm, 5 min), and nucleic acids were collected from the lysate by ethanol precipitation. The pellet was washed with 70% ethanol, and dissolved in 300 µl Low-TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8) containing 0.5 µg/ml RNase A (Sigma-Aldrich) to inhibit the remaining RNA. After 1 hour incubation at 37°C, DNA was quantified using the BioDrop μLITE (BioDrop, Cambridge, UK). Nextera kit
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: sampling time point
attributes.value: after 15 cycles of biofilm formation