Fig. 3

χ7122 pgl integrants express a functional N-glycosylation system. a Schematic representation of an χ7122 pgl integrant and its function; b western blotting of OD_600nm normalised periplasmic extracts showing the four χ7122 pgl clones tested are capable of decorating a heavily glycosylatable version of acceptor protein G-ExoA(10) with C. jejuni heptasaccharide, + ctrl consists of glycoengineering E. coli strain SDB1 transformed with a plasmid encoding the pgl locus (pACYCpgl) and a plasmid expressing L-arabinose inducible G-ExoA(10) (this latter present in all other samples);− ctrl consists of χ7122 wt transformed with G-ExoA(10)-encoding plasmid. Percentage in parenthesis indicates sample loading (v/v) per lane; c western blotting showing results of a His-pulldown from clarified lysates of the same samples shown in b; d densitometry analysis of b indicate χ7122 pgl integrants achieve ~ 4% glycosylation efficiency of G-ExoA(10) in comparison to + ctrl, * indicates p < 0.05 (two-tailed t-test); e dot-blotting of OD_600nm normalised cells washed and resuspended in PBS shows χ7122 pgl integrants maintain surface expression of their own O78 O-antigen, while no detectable surface expression of C. jejuni glycan is observed. + ctrl and – ctrl for O78-antigen detection are χ7122 wt and O-antigen negative E. coli W3110, respectively. + ctrl and − ctrl for C. jejuni glycan detection are W3110 constitutively expressing the pgl locus from pACYC plasmid and χ7122 wt, respectively. The four clones of χ7122 pgl tested are those verified by sequencing