RB-TnSeq of Novosphingobium aromaticivorans DSM12444: LB 2
An aliquot of the library was thawed and cultured in LB supplemented with 50mg/L kanamycin and grown overnight. The culture was diluted 1:100 into three flasks containing 2g/L glucose SMB minimal medium and grown to saturation. Samples were collected from each flask and the cell density was measured with a Klett-Summerson colorimeter. Each culture was then diluted to a starting cell density of 40 Klett units in SMB minimal medium with 1g/L glucose or 1g/L DC-A as the sole carbon source. The cultures were grown to saturation, split into 0.6mL aliquots, frozen, and stored at -80C. The cells were harvested by centrifugation (2,300 x g for 5 minutes) at 4C, resuspended in lysis buffer (0.16mM EDTA and 2% SDS), and incubated at 65C for 5 minutes. Genomic DNA was extracted using 25:24:1 phenol:chloroform:isoamyl alcohol. Barcode DNA sequences were amplified from the genome using custom indexing primers BarSeq_P1 and BarSeq_P2_IT001 to BarSeq_P2_IT009 (from Wetmore KM, Price MN, Waters RJ, Lamson JS, He J, Hoover CA, Blow MJ, Bristow J, Butland G, Arkin AP, Deutschbauer A. 2015. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6:e00306-15.)