, 2008) was used as a PCR template for amplification of the rpsL-neo cassette. Luria–Bertani (LB) medium and SOC medium were prepared as described elsewhere (Sambrook et al., 1989). All other bacteria were routinely grown in LB media at 37 °C unless stated otherwise. Antibiotics were added at the following concentrations for plasmid and/or

recombinants selection: click here ampicillin (Amp) (100 μg mL−1), kanamycin (Km) (50 μg mL−1), streptomycin (Strept) (100 μg mL−1), and tetracycline (Tet) (5 μg mL−1). Streptomycin-resistant (StreptR) derivatives of APEC1 (APEC 1-StrR strain) were obtained by serial culturing in increasing concentrations of streptomycin (50–150 μg mL−1) to facilitate isolation of the strains in subsequent experiments. DNA manipulations were performed as described elsewhere (Sambrook et al., 1989). Electrocompetent cells were prepared using standard procedures unless stated otherwise (Sambrook et al., Sirolimus 1989). Electroporation was carried out using Bio-Rad® Gene Pulser Xcell™ (Bio-Rad® Laboratories Inc., Richmond, CA) at 1.7 kV with 25 μF and 200 Ω. Plasmid DNA and DNA fragments were purified using commercial kits purchased from Fermentas (St. Leon-Rot, Germany). PCR amplifications were performed using AccuPrime™ Taq

High Fidelity polymerase (Invitrogen) or SuperTaq polymerase (SphaeroQ, Leiden, The Netherlands). Oligonucleotides were manufactured by Sigma-Aldrich (Bornem, Belgium). The PCR products were visualized on a 1% agarose gel containing SYBRsafe (Invitrogen) by transillumination. Smart ladder® (Eurogentec, Seraing, Belgium) was used as a molecular weight marker. Overnight bacterial cultures of APEC1-StrR were diluted 1 : 100 into 40 mL of fresh LB medium and incubated at 37 °C, 230 r.p.m. until they reached an OD600 nm of ~0.5–0.6. Cultures were then incubated on ice for 30 min

and then concentrated by centrifugation at 1700 g for 15 min at 4 °C. From this step, everything was maintained on ice. After discarding the supernatant, cells were then resuspended in 40 mL of ice-cold 10% glycerol Adenylyl cyclase and centrifuged again at 1700 g for 10 min at 4 °C. After repeating the washing steps four times, the cells were suspended in ice-cold 10% glycerol and stored in 20 μL aliquots in prechilled 1.5 mL microcentifuge tubes. The electrocompetent cells were either used immediately for electroporation or stored at −80 °C. Plasmid pKD46 encoding the lambda Red recombinase was transformed into APEC1-StrR by electroporation. Immediately after electroporation, cells were incubated for 2 h at 30 °C, 230 r.p.m. Five hundred microliters of the mixture was plated on LB-Amp, and the plates were incubated at 30 °C overnight. Ampicillin-resistant (AmpR) colonies were subcultured into LB-Amp broth and incubated at 30 °C for 8 h and subsequently stored in 15% LB-glycerol at −80 °C.