Fig. 3.

A) Randomly picked clones probed for production of the C. jejuni heptasaccharide using an SBA, a biotinylated GalNAc-specific lectin, and green streptavidin-coupled IRDye. Clones were selected at random, grown in high throughput 200-μL cultures, and spotted on nitrocellulose membrane. Clones selected for further characterization are circled in red. B) Clones were subsequently grown in 10-mL cultures, normalised to the same OD, and probed for GalNAc production with SBA. Cells harboring plasmid-borne native unmodified pgl cluster and empty plasmid control are denoted as “+” and “-,” respectively. C) ELISA glycan quantification shows a range of glycan production from refactored clusters compared with the wildtype cluster. D and E) Glycoconjugate produced by 8 refactored pgl cluster variants from 2 independent assemblies and transformations. Glycoproteins were resolved by SDS-PAGE and were detected by immunoblot analysis probed with anti-6xHis antibody (red) and SBA lectin (green). Coordinates corresponding to position of clones in the 96 well plate are denoted on the blots.