Concentrator 5301

Lipopeptide-rich fraction (LRF) from the liquid culture medium of bacteria was obtained using ethanol extraction [23 (link),25 (link)]. After completion of cultivation, the bacterial suspension was centrifuged at 4000× g at 4 °C for 30 min in an Avanti J-E centrifuge (Beckman Coulter, Bray, OK, USA), the supernatant was acidified with by adding 2 M HCl to pH 2.0 and incubated overnight at 4 °C. The formed precipitate was washed with distilled water acidified to pH 2.0 with 2 M HCl and centrifuged twice at 4000× g for 30 min. The resulting precipitate was extracted twice with 80% ethanol (pH 7.0). The crude extract was purified using an Amicon Ultracel—3K filter (Merck KGaA, Darmstadt, Germany), the fraction with a molecular weight of less than 3 kDa was collected and dried on a vacuum concentrator (Eppendorf Concentrator 5301, Eppendorf, Hamburg, Germany) at 30 °C. The dried residue was weighed and subsequently re-dissolved in 80% ethanol and different concentrations were used in the experiments.

The samples were collected as mentioned above for assessment of essential oil at 0, 1, 2, 3, 4, 5, and 6 weeks, respectively. For each sampling, five replicate stems were used for assessment of essential oil with each treatment (a total of 140 stems). The samples were immediately immersed in liquid nitrogen and ground to a powder with a pestle and mortar. For each treatment, 1 g powdered stem samples were weighed, immersed in 30 ml petroleum ether and shaken for 24 h. After filtration and concentration (Concentrator 5301, Eppendorf, Germany), essential oil was obtained and the oil content was also calculated.
Essential oil from the 6 week harvest was used for GC-MS with an Agilent 6890 N-5975 I system with an Innowax DB-5MS column (30 m × 0.25 mm, 0.25 μm film thickness). Helium was used as carrier gas at a flow rate of 1 mL min^-1. Oven temperature was programmed to 70°C for 1 min, raised to 250°C at a rate of 8°C min^-1, and kept constant at 250°C for 15 min. Mass spectra were recorded at 70 eV with the mass range m/z 35 to 450. The identification of essential oil components was done by computer matching against NIST and Wiley GC-MS Library or comparing the retention times of oil components with standard samples.

The gut microbiota analysis was performed as described in Prochazkova et al. (24 (link)). Briefly, total DNA was isolated from stool samples using the ZymoBIOMICS DNA Miniprep kit (Zymo Research). The primer set with barcodes (342F/806R) was used to amplify the V3-V4 region of the 16S rRNA gene using KAPA HiFi HotStart ReadyMix (Roche). Triplicates of amplicons were pooled, normalized with the SequalPrep^TM Normalization Plate Kit (ThermoFisher Scientific), concentrated (3 h, 30°C under vacuum; Concentrator 5301, Eppendorf), purified (DNA Clean & Concentrator Kit, Zymo Research), and ligated with sequencing adapters (TrueSeq DNA PCR-free LT Sample Preparation Kit, Illumina) using the KAPA HyperPlus kit (Roche). The final libraries were pooled in equimolar concentrations and sequenced on an Illumina Miseq using the Miseq reagent Kit v3 (Illumina). The bioinformatic pipeline is described in the Supplementary Materials.

Protein spots of interest were cut from the gels to be destained with 100 μL of 50% (v/v) acetonitrile (ACN) in 25 mM ammonium bicarbonate for 1 h. Then an aliquot (3 μL) of trypsin solution (10 μg/mL; Amresco Inc., Solon, OH) in 25 mM ammonium bicarbonate was added to each gel piece after it was completely dried by vacuum centrifugation (Eppendorf Concentrator 5301, Eppendorf, Hamburg, Germany) for 30 min. The mixture was incubated in a trypsin solution at 4°C for rehydration for 1 h, followed by incubation at 37°C for 12 h. Thereafter, the gel pieces were vacuum-dried to evaporate the solvent and 8 μL of 5% trifluoroacetic acid (TFA) was added onto the dry gel pieces. After incubation at 37°C for 1 h, the solution was transferred into a microcentrifuge tube. The gel pieces were extracted twice separately with 8 μL of 2.5% TFA and 50% acetonitrile, and 5% TFA. The third extraction was performed using 8 μL of 100% acetonitrile and the solution was combined with the previous two extracts in the microcentrifuge tube. Finally, the combined solution was dried and resolubilized in 2 μL of 0.5% TFA for protein identification using MALDI-TOF/TOF MS (Matrix Assisted Laser De sorption Ionization-Time of Flight/Time of Flight MS).

In order to investigate the capability of the assay to distinguish between target and non-target, DNA from 10 isolates of E. coli, 5 isolates of Enterococcus spp., 6 isolates of Salmonella spp., 3 isolates from Vibrio spp., and 7 isolates of V. cholerae were used as templates. The concentration of all DNA samples from the isolates was kept almost same (approximately 10 ng/μL) by diluting with DEPC-treated water or concentrating by DNA concentrator (Eppendorf Concentrator 5301).

The strains were cultivated in tryptic soy broth (TSB) at 37°C with linear shaking at 100 rpm in a water bath (OLS200, Grant Instruments, England). Strains were grown in two sets. In the first set the wild type and the ∆sigB, ∆agr, ∆sarA and ∆agr/∆sarA mutants were cultured and in a second set the wild type and the ∆sigB∆agr, ∆sigB∆sarA and ∆sigB∆agr∆sarA mutants were cultured. The wild type always served as control. During exponential growth phase bacteria were pelleted by centrifugation and culture supernatants were mixed with 10% final concentration of TCA and proteins precipitated at 4°C overnight. Pelleted proteins were washed five times with 70% ethanol and then incubated for 30 min at 21°C and mixed at 600 rpm in a thermomixer (Eppendorf, Germany). Afterwards, pellets were washed once with 100% ethanol and dried in a speed vacuum centrifuge (Concentrator 5301, Eppendorf, Germany). Subsequently, protein pellets were dissolved in a suitable volume of 1x UT buffer (8 M urea and 2 M thiourea) and incubated for 1h at 21°C with shaking at 600 rpm in a thermomixer (Eppendorf, Germany). No soluble components were pelleted via centrifugation. Protein concentration was determined according to Bradford [71 (link)]. 4 μg of protein were reduced and alkylated with Dithiothreitol and Iodoacetamid prior to digestion with Trypsin.

To prepare working solutions of Aβ₄₂ and pS8-Aβ₄₂ peptides, cold hexafluoroisopropanol (“Fluka”) was added to dry Aβ to a concentration of 1 mM and incubated for 60 min at room temperature. The resulting solution was then placed in ice for 10 min and transferred to non-siliconized microcentrifuge tubes (0.56 mg peptide per tube). The peptides in the tubes were vacuum-dried with Eppendorf Concentrator 5301. The resulting dry peptides were stored at −80°C. A fresh 2.5 mM Aβ solution was prepared by adding 20 μl of 100% anhydrous dimethyl sulfoxide (“Sigma”) to 0.56 mg of the peptide, followed by incubation for 1 h at room temperature.