Excessive-throughput depletion experiments
Every pressure was inoculated from strong R2A plates on a 96 effectively plate in organic triplicates and 5 technical replicates in a medium supplemented with 20 amino acids and 4 nutritional vitamins: thiamine, niacin, biotin, and pantothenate at 200 µl tradition quantity. Important cultures had been ready subsequent day by diluting 10 µl of every cell tradition into recent media. After an in a single day tradition, late exponential stage was reached. The 5 technical replicates had been pooled collectively right into a sterile 2 ml Eppendorf tube and washed twice by centrifuging the cell suspension tube (10,000 rpm, 2 min), eradicating supernatant, and dissolving the cell pellet in 1.5 ml of sterile MgCl2. After the second wash, cells had been collected by way of a centrifugation step as earlier than, and the ensuing pellets had been dissolved in 50 µl of MgCl2. 5 µl of the ensuing cell suspension was inoculated in 195 µl of 5 media that contained all 20 amino acids and differed in vitamin dietary supplements. One of many media was a management medium, and it included all 4 nutritional vitamins because the preculture medium. The opposite 4 media every lacked one of many following nutritional vitamins: thiamine, biotin, niacin, or pantothenate. Development was then monitored on a Tecan plate reader (for particulars see “Development assays”). After each 2 doublings, cultures had been diluted as acceptable to take care of exponential development within the plate reader’s linear vary.
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