Yeast batch cultures for production of metabolites were performed with the System Duetz (EnzyScreen, Heemstede, Netherlands). All incubation steps for growth and extraction were performed in an ISF-1-W shaker (Kuhner, Birsfelden, Switzerland) at 30 °C, 300 RPM, and 5 cm shaking diameter. Precultures were inoculated in a square 96-half-deepwell microplate from a single colony in three replicates in 300 μl SC dropout medium, as required for plasmid selection, and incubated for 24 h. Optical density at 600 nm (OD600) of a 1:50 dilution was measured in an Ultrospec 10 table top spectrophotometer (GE Healthcare, Little Chalfont, United Kingdom). Main cultures were inoculated in 2 ml of the same SC medium in a 24-deepwell microplate to an OD600 of 0.1 and incubated for 72 h. Final OD600 of a 1:50 dilution was measured and 300 μl broth was extracted with 300 μl methanol by incubating for 10 min in a 96 square deepwell microplate and finally clarified by centrifugation at 4000g for 5 min.
Ultrospec 10
Manufactured by GE Healthcare
Sourced in United Kingdom
The Ultrospec 10 is a compact and robust UV-Visible spectrophotometer designed for routine analysis. It provides accurate and reliable measurements in the wavelength range of 340 to 800 nm. The instrument is easy to use and can be operated with minimal training.
Automatically generated - may contain errors
Lab products found in correlation
Epoch2 spectrophotometer, by Agilent Technologies (1 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Trypticase soy, by bioMérieux (1 mentions)
Sheep blood plate, by bioMérieux (1 mentions)
Rotorgene rg 2000, by Qiagen (1 mentions)
Rnasin rnase inhibitor, by Promega (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Dnase 1, by Roche (1 mentions)
Rnaprotect bacteria reagent, by Qiagen (1 mentions)
8 protocols using ultrospec 10
1
Yeast Batch Culture Metabolite Production
2
Tracking Piscirickettsia salmonis Growth
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100 µL aliquots of deep-frozen P. salmonis wild-type and mutant strains were streaked onto PSA plates and incubated for 2 days at 18°C. By this time, a homogeneous bacterial lawn had formed and we proceeded to suspend a loopful of bacteria in 1 mL of buffered saline solution (BSS: 8.5 g/L NaCl, 2.0 g/L K2HPO4, 2.0 g/L KH2PO4, pH 6.4), transferred them onto new PSA plates, and incubated as mentioned above. Bacterial suspensions (OD600 = 1.0) were prepared by mixing bacteria with 5 mL of cold BSS in a conical tube kept at 10°C. For growth kinetics, 96-well microplates were inoculated with 150 µL/well of 1:100 bacterial suspensions in PSB and incubated during 70 h. Hourly reads were realized with an EPOCH2 spectrophotometer (Biotek, USA). By contrast, self-agglutination tests lasted 8 h, but used bacterial suspensions prepared in PSB that were held in plastic cuvettes at 18°C and pH 7.2. Reads were collected each hour with a densitometer (Ultrospec 10, GE LifeSciences). In order to determine bacterial counts, aliquots of log10 dilutions in PSB were seeded on PSA plates in triplicates and incubated at 18°C for one week. Acriflavine tests were carried out using fresh bacterial suspension in BSS mixed with identical volumes (10 µL) of 0.1% acriflavine on a glass slide.
3
Standardized MRSA Strain Preparation
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Methicillin-resistant S. aureus (MRSA) strain LUH14616 (NCCB100829) stocks were stored in 20% glycerol at −80°C until use. Prior to experiments, stocks were thawed and spread on trypticase soy agar with 5% sheep blood plates (Biomerieux, Marcy-l’Étoile, France, 43009) and cultured aerobically overnight at 37°C. Thereafter, bacteria were cultured aerobically at 37°C to mid-log phase in tryptic soy broth (Oxoid, Basingstoke, Hampshire, UK, CM0129) for 2.5 h under continuous spin at 200 rotations per minute. Bacteria were concentrated by centrifugation at 1,000×g for 10 min and washed with phosphate-buffered saline (PBS, pH 7.4). Ultimately, the bacteria were resuspended in brain heart infusion (BHI) broth (Oxoid, Basingstoke, Hampshire, UK, CM1135B) to the required concentrations based on the optical density at 600 nm, measured using Ultrospec 10 (GE Healthcare Bio-Sciences AB, Cambridge, UK).
4
RNA Extraction and Real-time RT-PCR for S. pneumoniae
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The liquid culture was grown at 37 °C to an OD600 = 0.4 determined using a cell density meter (Ultrospec 10, GE Healthcare formerly Amersham Biosciences). Total RNA was immediately stabilized with RNAprotect bacteria reagent (QIAGEN, Valencia, CA) and then extracted from S. pneumoniae strains by using a Qiagen RNeasy kit according to the manufacturer’s instructions. RNA was resuspended in nuclease-free water in the presence of RNasin RNase inhibitor (Promega). Contaminating DNA was digested with DNase I (Roche Diagnostics). Real-time RT-PCR was performed on a Rotorgene RG-2000 (Corbett Research, Mortlake NSW, Australia) and as described elsewhere5 (link). Briefly, the cDNA (20 ng/reaction) was used for real-time amplification using the primers listed in Table S2 . The mRNA level of 16S rRNA was used as an internal control in order to normalize all data. The amounts of transcripts were expressed as the n-fold difference relative to the control gene (2−ΔCT, where ΔCT represents the difference in threshold cycle between the target and control genes).
5
Biomass and Metabolite Quantification
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Biomass formation was measured by determination of the OD600 (Ultrospec 10, GE Healthcare, USA) at specific time points. The cell dry weight (gCDW/L) was correlated to the OD600 in several independent cultivations with a correlation factor of 0.346 gCDW/L per OD (data not shown). Shaking flasks were sampled directly in the incubator using an injection syringe (100 Sterican®, 0.80 × 120 mm, B.Braun, Melsungen, Germany). For the determination of isobutanol, 2‐KIV, 2‐ketogluconate (2‐KG), and glucose concentrations, 2 mL of the main culture was harvested by centrifugation (12 100 × g, 5 min, room temperature (RT)) and the supernatant was analyzed via HPLC. Glucose concentrations were measured enzymatically with a test kit from r‐biopharm (r‐biopharm AG, Darmstadt, Germany).
6
Culturing P. aeruginosa Strains for Experiments
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We used the P. aeruginosa strains PA103∆exoUexoT::Tc pUCPexoY (“ExoY”) and PA103∆exoUexoT::Tc pUCPexoY-K81M (“ExoYK81M”). Bacteria were stored as glycerol stocks at −80 °C. Bacteria were streaked on Vogel Bonner (VB) medium agar plates containing 400 µg/mL carbenicillin and incubated at 37 °C for 16 h. The next day, a large loopful of bacteria was suspended in PBS and the number of colony forming units (cfu)/mL was determined by measuring the optical density at a wavelength of 600 nm (OD600) with the Ultrospec 10 cell density meter (GE Healthcare, Buckinghamshire, UK), OD600 = 0.25 ≈ 2 × 108 cfu/mL. Desired bacterial concentrations were adjusted by serial dilution with PBS and checked on VB dose control plates. A brief description of the bacterial strains used in this study is shown in Table 2 .
7
Quantification of Biomass and Metabolites
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The biomass concentration was measured with a spectrophotometer (Ultrospec 10, GE Healthcare Life Sciences). In this device the OD600 correlates to cell dry weight (CDW): 1 OD600 = 0.505 gCDW L-1. The samples taken during cultivation were centrifuged at 13,300 rpm for 3 min and stored at -20°C for further analysis. To follow the consumption of the glucose and derivatives (gluconate and 2- ketogluconate) by the P. putida KT2440 strains, a Beckman HPLC equipped with an organic acid resin column (polystyrol- divinylbenzol copolymer, PS-DVB: 300 × 8.0 mm, CS-Chromatographie) was used with 5 mM H2SO4 as eluent at a flow of 0.8 mL h-1 for 11 min at 75°C. Detection was realized with an UV detector at a wavelength of 210 nm and a RI detector. The oAB production was analyzed with a reverse phase column (LiChrosorb 100 RP-18, 250 × 4 mm, Merck), at a flow of 1.2 mL h-1 [pump gradient of H2O + 0.1% TFA (pump A) and of MeOH (pump B): 0–2 min 90% A, 2–12 min gradient 0–90% A, 12–14 min 0% A, 14–15 min gradient 0–90% A, and 15–16 min 90% A] at 30°C. Detection was realized with an UV detector at a wavelength of 257 nm and a RI detector.
8
Stress-induced long-term live imaging
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We employed sub-lethal stress conditions to allow long live cell imaging sessions. Oxidative stress and acidic shift stress were induced by adding, respectively, 0.6 mM of H2O2[2] (link) and 150 mM of MES to the culture in exponential phase. Upon addition of MES, the pH very quickly shifts from 7.0 to 5.0 [12] (link). To confirm if stress responses were induced, we measured cell growth rates from the absorbance at OD 600 nm with a Spectrophotometer (Ultrospec 10, GE healthcare), every 30 minutes up to 4.5 hours (see File S1 ).
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