Illumina NovaSeq 6000 paired end sequencing; Experimental evolution of Pseudomonas aeruginosa biofilms exposed to furanone C-30 with and without tobramycin
Experimental evolution of Pseudomonas aeruginosa biofilms exposed to furanone C-30 with and without tobramycin
The biofilm is treated for 24h with tobramycin and C-30 alone or a combination of both for 24h. A sample of the treated biofilm is inoculated in LB and incubated overnight. The DNA-extraction is performed on 3ml of this overnight culture with OD590 = 2. P. aeruginosa PAO1 biofilms were grown in the synthetic cystic fibrosis sputum medium (SCFM2) for 24h. The biofilm was grown in a flat bottom 96-well plate and the inoculum was approx. 5 x 107 CFU/ml. P. aeruginosa biofilms were treated with tobramycin, furanone C-30 or a combination of both in fresh SCFM2 to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. A sample from this biofilm was used to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. A sample from the treated biofilm was regrown overnight in LB and 3 ml of a culture with OD590 = 2 was resuspended in 200 µl Tris- EDTA buffer (1mM EDTA, 10 mM Tris-HCl, pH 8.5; Gibco). 100 µl of this culture was added to 500 µl of lysis buffer (50 mM Tris-HCl pH 8, 70 mM EDTA pH 8, 1% sodium dodecyl sulfate, Sigma), containing 0.5 µg/ml pronase (Roche, Mannheim, Germany) and acid washed glass beads (Sigma). After vigorous vortexing for 10 s, the samples were incubated at 37°C during 4 min. Next, 400 µl of saturated ammonium acetate was added and the samples were vortexed for 10 s prior to centrifugation (2 min, 13000 rpm). Subsequently, 600 µl of chloroform was added and the samples were vortexed for another 10 s. The phases were separated by centrifugation (5 min, 13000 rpm) and 400 µl of the top aqueous phase was transferred to a DNAse free Eppendorf tube (Lobind tube, Eppendorf, Aarschot, Belgium) containing 1 ml of 100% ethanol. After centrifugation (5 min, 13000 rpm) the pellet was washed with 500 µl of 70% ethanol and dried in a SpeedVac (Thermo Fisher Scientific, USA) for 10 min. Finally, the extracted DNA was dissolved in 100 µl of low EDTA- Tris buffer (0.1 mM EDTA, 10 mM Tris-HCl, pH 8.5) containing 0.5 µg/ml RNAse (Qiagen, Venlo, The Netherlands) and was incubated at 37°C for 1 h. The DNA was subsequently quantified using a BioDrop µLITE (BioDrop, Cambridge, UK). NEB prep kit