IC50 is #selleck chemical randurls[1|1|,|CHEM1|]# the concentration that reduces the viability of the cells by 50%. Generation of resistant mutants against vz0825 The protocol for the generation of resistant mutants was the same as used in the publication of Bielecki et al. . V. cholerae strain NM06-058 was plated at a cell
number of 1 × 109 CFU on LB agar plates containing 8 μM vz0825 (5-times the MIC value). After incubation for 24 h at 37°C, micro-colonies were visible. 15 colonies were picked and preserved as mutants against vz0825. Isolation of genomic DNA and sequencing of genome-pool Isolation of the genomic DNA was performed according to the protocol of the DNeasy Blood and Tissue Kit (Qiagen). Briefly, the 15 resistant mutants were inoculated individually in 5 ml LB medium and incubated for 6 h at 37°C with shaking at 180 rpm. In parallel, the wild type strain was cultivated under identical conditions. Based on the click here OD600 measurements of the cultures, the 15 mutants were pooled in equal amounts. After adjusting the cell number at 2 × 109 CFU the pooled mutants and the wild type strain were collected by centrifugation. The cell pellets were lysed by addition of ATL buffer
and proteinase K for 1 h at 56°C. RNA was removed by addition of 4 μl RNase A (100 mg/ml) and incubation for 2 min at RT. 200 μl AL buffer and afterwards 200 μl of ethanol were added with mixing. The mixture was transferred
to DNeasy Mini spin columns and centrifuged at ≥ 6.000 × g for 1 min. Washing was carried out with 500 μl AW1 buffer followed by centrifugation for 1 min. A second washing step was carried out with 500 μl AW2 buffer. The tubes were centrifuged for 3 min at 20,000 × g and the genomic DNA was eluted from the membranes with 200 μl AE Arachidonate 15-lipoxygenase buffer. Whole genome sequencing, alignment and annotation were carried out in the sequencing facility of the HZI (head Dr. Robert Geffers). Libraries of DNA fragments with an average length of 300 bp were prepared according the manufacturer’s instructions “Preparing Samples for Sequencing Genomic DNA” (Illumina). Sequencing was carried out with the Illumina Cluster Station and the Genome Analyzer IIx. The resulting data was transformed into FastQ-format. Sequencing of the DNA library resulted in a total base count of 855,825,664 and 2,546,713,435 for wild type and resistant mutants genome pool, respectively. This corresponds to a calculated average coverage of 214 for the wild type and for each resistant mutant to a coverage of 42. The published complete genome has a total base number of 4,033,460 (Table 6, ). The sequencing procedure resulted in 11,260,862 and 35,196,596 reads for wild type and resistant mutants genome pools, respectively, which were mapped to the reference genome of the annotated V.