title: NextSeq 500 sequencing; TraDIS-Xpress to determine E. coli response to Piperacillin and Tazobactam separately and in combination
design: TraDIS-Xpress to determine E. coli response to Piperacillin and Tazobactam separately and in combination
library_construction_protocol: The E. coli BW25113 transposon mutant library used in this study contains over 800,000 distinct mutants and has recently been described by Yasir et al. (2020). Approximately 107 CFU/mL of this library was added to 1 mL LB broth supplemented with concentrations of piperacillin around the MIC (MIC was 4 µg/mL piperacillin, concentrations used were ¼x, ½x, 1x and 2x MIC). Tazobactam was either added or omitted from these conditions at a concentration of 32 µg/mL. The Tn5 transposon used to create this library contains an outward-transcribing tac promoter, which was either induced with 0.2 or 1 µM IPTG or left uninduced. Each condition was carried out in duplicate with two unstressed controls containing no drug. Cultures were grown for 24 hours at 37 ⁰C shaking and were centrifuged at 3000 x g for 10 minutes to pellet the cells DNA was extracted from cell pellets following the protocol described by Trampari et al. (2021) and quantified using a Qubit HS assay kit (Invitrogen).  The library was fragmented with the MuSeek DNA fragment library preparation kit (ThermoFisher) and purified using AMPure XP beads (Beckman Coulter). Fragments containing the transposon were amplified by PCR using customised biotinylated sequencing primers specific for the transposon tag and the PCR product was again purified as previously described. To extract biotinylated fragments containing the Tn5 transposon sequence, samples were incubated for 4 hours with activated beads from the Dynabeads® kilobaseBINDER™ kit (Invitrogen). Following this, Dynabeads® were resuspended in elution buffer and used as the template for a second PCR amplification step, using customised primers specific for the transposon and those specific for the MuSeek tag. Dynabeads® were removed from the PCR product using a magnetic stand. The resulting sequencing library was purified twice and fragments were selected between 300-500 bp in length.