In both cases the orientation of the antibiotic resistance cassette was the same as that of the target gene to avoid a negative polar effect in the mutants. Mutagenesis using the constructed derivatives was conducted via electroporation and selection of the derivatives on media supplemented with appropriate antibiotics. Allelic replacement was confirmed by PCR. The mutants were designated LCZ696 molecular weight 11168H/peb3::kan

r and 11168H/jlpA::cam r . Table 2 Primers Erastin price used for mutation of peb3 and jlpA and for complementation of peb3 Primer Sequence (5′-3′) Used for peb3_for ATGAAAAAAATTATTACTTTATTTGGTGCATG Mutation of peb3 gene peb3 _rev TTATTCTCTCCAGCCGTATTTTTTAAAAATTTC Mutation of peb3 gene jlpA_for ATGAAAAAAGGTATTTTTCTCTCTATTGG Mutation of jlpA gene jlpA_rev

TTAAAATGACGCTCCGCCCATTAACATAG Mutation of jlpA gene peb3_XbaI_for ATAATCTAGAAAGGAAATACTATGAAAAAAATTATTACTTTATTTGGTGC selleck chemical Complementation of peb3 mutation Peb3_XbaI_rev AGGTTCTAGATTAATGATGATGATGATGATGTTCTCTCCAGCCGTATTTTTTAAAAATTTC Complementation of peb3 mutation Complementation of peb3 mutant Peb3 gene was PCR amplified using primers described in Table 1. The product was digested with XbaI enzyme and cloned into XbaI-digested pRRC plasmid to produce pRRC_peb3. Restriction analysis verified that the gene was transcribed in the same orientation as the cam r gene. After transformation of the 11168H/peb3::kan r mutant with plasmid pRRC_peb3, KanrCamr clones were selected. PCR analysis confirmed integration of peb3 gene into one of the rRNA gene clusters. The complementation derivative was designated 11168H/peb3::kan r /peb3 + . Binding assay Bacterial attachment was studied in ELISA-like assay using a 96-well microtiter plate Maxisorp™ (Thermo Scientific) coated Soya Bean Agglutinin (SBA) lectin (Sigma) in bicarbonate-coating buffer: 5.3 g/L Na2CO3, 4.2 g/L NaHCO3, 1 g/L sodium azide, pH 9.6. Microtiter plate wells were incubated

overnight Immune system with SBA lectin (10 μg/ml) at 4˚C, followed by blocking with 1% Bovine Serum Albumin (BSA) overnight at 4°C. BSA-coated, wells were used as negative control. Bacteria (two-day cultures of C. jejuni or one-day cultures of E. coli) were harvested, resuspended in Phosphate-Buffered Saline (PBS) to OD600 = 1, 0.1 ml suspensions (corresponding to 4×108 c.f.u. of C. jejuni) were added to each well of the microtiter plate, followed by incubation for 40 min at room temperature. After rinses with PBS, supplemented with 0.2% Tween (PBST) the plate was incubated with biotinylated SBA lectin (Vectors Laboratories) for 60 min at room temperature. The wells were then treated with horseradish peroxidase-conjugated streptavidin (Sigma) for 30 min at room temperature followed by incubation with TMB (3,3′,5,5′-Tetramethylbenzidine) substrate (Sigma) for 10 min. The reaction was stopped by adding stop solution (1 M H2SO4). Binding was monitored by measuring OD at 450 nm.