The loxP sites in primers were designed in such a way that they were in unidirectional orientation in the cassette (Fig. 1a). Primers PS-341 research buy HT51 (F&R) and HT53 (F&R) were used to amplify the rpsL-neo cassette to generate loxP-rpsL-neo-loxP cassette flanked by homolog regions to intergenic region 2051–52 (Fig. 1b)

and fiu gene, respectively. The PCR conditions were 94 °C for 2 min (initial denaturation) then through 30 cycles of [94 °C for 30 s (denaturation), 62 °C for 30 s (annealing temperature), and 68 °C for 1 min and 30 s (elongation)], followed by a final elongation for 10 min at 68 °C. A single fresh colony of APEC1-StrR strain containing pKD46 was placed into 40 mL of LB-ampicillin and shaken at 30 °C in a 125-mL flask for 3 h and thereafter l-arabinose was added to a final concentration of 100 mM. At an OD600 nm of ~0.5–0.6, the cells were made electrocompetent following a protocol explained above. l-Arabinose was used for the induction of the lambda Red genes expression. The PCR DNA/RNA Synthesis inhibitor products (approximate size 1.4 kb) obtained were purified, digested with

DpnI (Fermentas, Germany) to remove the template plasmid, re-purified and suspended in nuclease-free water. Approximately 0.1–0.3 μg of PCR products were mixed with electrocompetent cells (APEC1-StrR strain containing pKD46) and electroporated. The mixture was incubated for 3 h at 37 °C, in a shaking incubator (100 r.p.m.) and 500 μL were plated on LB-Km, incubated at 37 °C overnight. The kanamycin-resistant (KmR) colonies obtained were colony purified nonselectively at 37 °C and then grown at 43 °C to cure the plasmid pKD46. Confirmation of the loss of the plasmid was carried out by ampicillin sensitivity test. Integration of the LoxP

Fossariinae cassette at the correct position on the bacteria chromosome was confirmed by three colony PCRs. One PCR was carried out using primers flanking the integrated region while the other two PCRs were carried out using locus-specific primers. Freshly isolated colonies were touched each by a separate sterile plastic tip and resuspended into 2 μL of sterile Milli-Q water. The PCR conditions were as follows: 94 °C for 5 min (initial denaturation), then through 35 cycles of [94 °C for 30 s (denaturation), different annealing temperatures depending on primer set for 30 s (annealing) and 72 °C for 2 min (elongation)], followed by a final elongation at 72 °C for 5 min. The PCR products were visualized on a 1% agarose gel. The marker flanked by loxP sites was deleted using Cre recombinase, which recombines the two loxP sites resulting into deletion of the flanked piece. KmR strains were transformed by a temperature-sensitive plasmid pSC101-BAD-Cre-tet, which contains the cre gene tightly regulated by a PBAD promoter (induced by l-arabinose) and incubated at 30 °C overnight.