Co-culture experiments
As a pre-experiment, CFU counts per unit of OD had been decided utilizing a dilution plating technique. Colonies had been counted from a dilution that allowed for willpower from 5 µl dots (Supplementary Desk 5). All 20 strains had been then inoculated from strong R2A plates into liquid medium containing 20 amino acids and 10 nutritional vitamins and grown in a single day in organic triplicates in 10 ml tradition quantity. After the pre-culture, all strains had been in stationary section. The OD of every tradition was measured and adjusted to 5e8 cells per ml. These adjusted cultures had been then mixed into an inoculum the place every pressure had an abundance of roughly 1/20. The inoculum was washed as described in part “Pattern preparation for LC/MS evaluation of depletion experiments”. 5 sequencing samples had been taken from every inoculum, and three technical replicates in each vitamin-supplemented and vitamin-free media had been inoculated from every inoculum. Tradition volumes had been 20 ml, and cultures had been allowed to develop at 28 °C with 200 rpm orbital shaking. At 9, 24, 48, 72, 96, and 120 h, samples for 16S rRNA gene sequencing and exometabolomics had been taken as follows. For 16S rRNA gene sequencing, a 1 ml pattern was transferred to a DNA extraction tube from FastDNA SPIN Package for Soil. The tubes had been centrifugated (10,000 rpm, 5 minutes, 4 °C), supernatants had been eliminated, and the tubes with cell pellets had been frozen and stored at -80 °C till extraction. For exometabolomics, 1 ml of tradition was transferred to a 2 ml Eppendorf tube and centrifugated (10,000 rpm, 5 min, 4 °C). 200 µl of supernatants had been saved in two technical replicates on a 96 effectively plate and saved at −20 °C till evaluation (see “LC/MS evaluation” for particulars).
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