A single-colony of each strain was grown in 5 mL of YNB supplemented with d -glucose in a 50 mL conical tube (Sarstedt AG & Co., Nümbrecht, Germany) and incubated at 30 °C and 180 rpm. At the end of the exponential growth phase, the cultivations were stopped and the amount of cells was normalized by dilution with 0.9% NaCl to an OD620 of 2.5, which corresponds to approximately 107 colony forming units (CFU) per mL. Serial dilutions of each strain were done under sterile conditions with 0.9% NaCl to obtain a coverage from 105 to 108 CFU mL−1. 5 µL of each dilution were carefully dot plated in YNB-agar plates supplemented with d -glucose (5 g L−1), d -xylose (20 g L−1) or a mixture of d -glucose/d -xylose (5 and 20 g L−1, respectively) and incubated at 30 °C for 48 h.
50 ml conical tube
Manufactured by Sarstedt
Sourced in Germany, Canada, United States
The 50-mL conical tube is a laboratory equipment used for various applications in scientific research and testing. It is a commonly used container for holding and transporting liquid or solid samples. The tube has a conical shape that facilitates sedimentation and centrifugation of samples.
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6 protocols using 50 ml conical tube
1
Yeast Growth on Glucose and Xylose
2
Tracking P. aeruginosa Growth Dynamics
3
Bacterial Catalase Activity Assay
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Bacterial strains were grown overnight at 37 °C with 250 rpm shaking in LB broth into lag phase prior to the assay in a total volume of 5 mL in a 50 mL conical tube (Sarstedt, Montréal, QC, Canada). Absorbance at 600 nm was measured prior to the assay for the purpose of normalization of the catalase activity based on the growth of each strain. The catalase activity assay was carried out using a Gilson oxygraph equipped with a Clark electrode as described previously [72 (link)]. The assay was carried out in biological duplicates and the average values with the standard deviations were plotted and statistically analyzed using GraphPad Prism v6.07 (La Jolla, CA, USA).
4
Fungal Growth Cultivation Protocol
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Vogel's media (50 mL, 3% glucose w/v) in a 250-mL Erlenmeyer flask was inoculated under aseptic conditions with conidial suspension to a total concentration of 2.7 × 104 cells/mL (21 μL conidial suspension), and covered with aluminum foil. Liquid cultures were grown with orbital shaking (~237 rpm) at room temperature (~25°C) under constant cool white light (7 μmol L−1 s−1 m−2) for 32 h. At that point mycelia consistently formed a single, cohesive mass. Mycelia for 13C glucose experiments were allowed to grow for 48–52 h. The entire culture was transferred to a 50 mL conical tube (Sarstedt; Newton, NC, USA) for transport to the NMR facility (15–30 min).
5
Comparing Growth Kinetics of P. aeruginosa Strains
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Glycerol stocks of P. aeruginosa WT and G2L4 RT knock-out strains were streaked on LB agar and incubated at 37˚C overnight. The next day, a single colony was inoculated into 5-mL LB medium in a 50-mL conical tube (Sarstedt) and incubated at 37˚C overnight with shaking (200 rpm). A 1-mL aliquot of the overnight culture was then added to 100-mL LB in a 250-mL Erlenmeyer flask and incubated at 37˚C with shaking (200 rpm). 0.5-mL samples of P. aeruginosa WT and G2L4 RT KO cultures were collected every 6 h for up to 72 h, serially diluted, and plated on LB agar. The plates were incubated overnight at 37˚C, and colonies were counted to calculate colony forming units (CFUs).
6
Genomic Analysis of P. aeruginosa Knockout Strains
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Glycerol stocks of P. aeruginosa WT and G2L4 RT knock-out strains were inoculated into 5-mL LB medium in a 50-mL conical tube (Sarstedt) and incubated at 37˚C for 16-18 h with shaking (200 rpm). The culture was then centrifuged at 4000 x g for 5 min, and genomic DNAs were extracted by using a Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's protocol. 1 µg of each genomic DNA was submitted to the Genome Sequencing and Analysis Facility (GSAF) at the University of Texas at Austin and sequencing libraries were prepared and sequenced on an Illumina MiSeq V2 instrument to obtain ~1 million 2
x 250 nt paired end reads per sample. Reads were mapped to the customized P. aeruginosa AZ-PAE12409 reference genome, which contains the targetron inserted at the designated location and the pBL1 vector used to express the targetron, using BWA with the default settings (Li and Durbin, 2010) . The genomic DNA coverage was calculated as mean coverage of 500-bp bins along the genomic sequences and plotted using R. Variants were called using freeBayes on bam files from genomic alignment of the WT or KO dataset, with the following settings: --ploidy 1 --min-mapping-quality 30 --min-alternate-count 10 ( Garrison and Marth, 2012) . The statistic test on KOspecific variants (point mutations) against the WT was analyzed by VarScan (Koboldt et al., 2009) .
x 250 nt paired end reads per sample. Reads were mapped to the customized P. aeruginosa AZ-PAE12409 reference genome, which contains the targetron inserted at the designated location and the pBL1 vector used to express the targetron, using BWA with the default settings (Li and Durbin, 2010) . The genomic DNA coverage was calculated as mean coverage of 500-bp bins along the genomic sequences and plotted using R. Variants were called using freeBayes on bam files from genomic alignment of the WT or KO dataset, with the following settings: --ploidy 1 --min-mapping-quality 30 --min-alternate-count 10 ( Garrison and Marth, 2012) . The statistic test on KOspecific variants (point mutations) against the WT was analyzed by VarScan (Koboldt et al., 2009) .
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