NextSeq 500 sequencing; Genome wide analysis of genes involved in efflux function and regulation within Escherichia coli and Salmonella enterica serovar Typhimurium
Genome wide analysis of genes involved in efflux function and regulation within Escherichia coli and Salmonella enterica serovar Typhimurium
The transposon mutant library in E. coli used in this study was described by Yasir et al. (2020) and contains over 800,000 unique mutants, which roughly equates to an insertion every 6 bp. The transposon mutant library in S. Typhimurium described by Holden et al. 2022 (in press) is also very dense, containing approximately 500,000 unique mutants and an insertion every 10 bp. Approximately 10^7 CFU/mL of transposon mutant library was added to 5 mL lysogeny broth (LB) in an untreated 6-well plate (Sigma). The minimum inhibitory concentration (MIC) of acriflavine in the parent E. coli and S. Typhimurium strains was determined to be 256 mg/L following the microbroth dilution method (EUCAST, 2021). Conditions were either left untreated or were supplemented with subinhibitory (64 mg/L) or inhibitory (256 mg/L) concentrations of acriflavine. Each condition was also prepared with or without 125 mg/L PAβN to elucidate genes involved in efflux specifically. Each condition was also prepared with and without 1 mM IPTG to induce expression from the transposon-located tac promoter, crucial for investigating how expression affect survival and for assaying essential genes. Two independent replicates were prepared for each condition. Plates were incubated at 30 ⁰C with light agitation at 60 rpm. Following 24 hours incubation, the cultures were centrifuged at 2100 x g for 10 minutes to form pellets, from which genomic DNA was extracted following the protocol described by Trampari et al. (2021) Genomic DNA was extracted following the protocol described by Trampari et al. (2021, NPJ Biofilms Microbiomes) Sequencing libraries were prepared following the protocol outlined by ( Yasir et al. (2020, Genom Res). Genomic DNA was fragmented using the MuSeek DNA library preparation kit (Invitrogen) and purified with 1.5x volume of AMPure beads (Beckman Coulter). Fragments containing the transposon were amplified by PCR using customised primers specific for the transposon and for the MuSeek tags. The PCR products were clean twice with 0.6x volume and 1x volume of AMPure beads to purify and size-select fragments between 300-500 bp.