organism: Pseudomonas aeruginosa PAO1
description: Protocols: 10 mL medium containing the dispersed cells was collected from the flow cells for each of the four conditions: cells that were released spontaneously (no dispersion trigger), glutamate dispersed cells, SNP dispersed cells, stop/start-flow dispersed cells and VNB dispersed cells. The remaining biofilms were collected from the flow-cells by dismantling the flow-cell and scraping the biofilm from the glass surface using a cell scraper, in 1 mL physiological saline. Pseudomonas aeruginosa PAO1 overnight cultures were cultivated in minimal medium which contained per liter: 2.56 g Na2HPO4, 2.08 g KH2PO4, 1 g NH4Cl, 0.04 g CaCl2x2H2O, 0.05 g MgSO4x7H2O, 0.06384 mg CuSO4, 160.23 ug ZnSO4x7H2O, 100 ug FeSO4x7H2O, 40 ug MnCl4x4H2O and 1.8 mmol glutamate. 5 mL of the overnight culture (OD600 = 0.05) was used to inoculate the flow-cell which was primed with the minimal medium. This was incubated for one hour at room temperature to allow the cells to adhere to the surfaces of the flow-cell. Next, the flow of minimal medium was started at a rate of 0.2 mL/min at room temperature for 96 hours and the flow cells further incubated at room temperature for 4 hours to form mature biofilms. Biofilm dispersion was induced on the four-day old biofilms by adding 18 mM glutamate or 500 uM Sodium NitroPrusside (SNP) to the medium flowing over the biofilms, by stopping the flow for one hour and restarting it (stop/start-flow) or by inducing vapor nanobubbles (VNBs). To induce VNBs 3 mL 70 nM gold nanoparticles (AuNPs, 1.4x1010/mL) were added to the biofilm and this was incubated for 15 minutes. Then, the AuNPs were washed off by allowing minimal medium to flow over the biofilm for 8 minutes. Then, the flow-cell was disconnected from the flow-setup to allow the formation of VNBs by a green pulsing laser (561 nm, 7 ns pulses, 1.69J/cm2). Each location of the biofilm received one laser pulse 20 mL of RNA-protect Bacteria Reagent (Qiagen) was added to the dispersed cells or 2 mL of RNA-protect Bacteria Reagent was added to the remaining biofilms. The samples were vortexed for 5 seconds and incubated for 5 minutes at room temperature. Then, the samples were centrifuged for 10 minutes at 5000 g. The pellet was resuspended in 100 uL TE-buffer containing 10 mM Tris, 1mM EDTA and 1 mg/mL lysozyme, pH 8. This was incubated for 15 minutes at and vortexed every 2 minutes for 10 seconds. From here on the protocol provided by the manufacturer of the RNeasy mini kit (Qiagen) was followed.The harvested RNA was DNase treated using Turbo DNA-free kit (Fisher Scientific) and the concentration of the RNA was determined with Quant-it Ribogreen RNA assay kit (Fisher Scientific). The ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit for Gram-negative bacteria (Illumina). The remaining RNA was purified using RNA clean & concentrator -5 kit (Zymo Research). The RNA was fragmented at 94C for 8 minutes in a 50:50 mixture of RNA and Elute Prime Fragment High Mix (Illumina). Library was prepared using Truseq stranded (m)RNA library preparation kit (Illumina). The libraries were quantified using qPCR, according to the Sequencing Library qPCR Quantification protocol guide (Illumina, version February 2011) and were pooled equimolarly.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-02-29T04:03:27Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-09-20T15:08:52Z
attributes.tag: External Id
attributes.value: SAMEA5790003
attributes.tag: INSDC center name
attributes.value: Laboratory of Pharmaceutical Microbiology, Ghent University
attributes.tag: INSDC first public
attributes.value: 2020-02-29T04:03:27Z
attributes.tag: INSDC last update
attributes.value: 2019-09-20T15:08:52Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8354:VNB dispersed 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: fraction
attributes.value: dispersed
attributes.tag: growth condition
attributes.value: flow-cell
attributes.tag: sample name
attributes.value: E-MTAB-8354:VNB dispersed 1
attributes.tag: scientific_name
attributes.value: Pseudomonas aeruginosa
attributes.tag: stimulus
attributes.value: biofilm dispersion by vapor nanobubbles
attributes.tag: strain
attributes.value: PAO1