Supplementary Figure 4: Effect of His6-WbdD expression level on the chain length of the E. coli O9a O-polysaccharide.
E. coli O9a ΔwbdD [pWQ470] cultures where grown to mid-exponential phase in LB medium containing 0.1% (w/v) D-mannose and varying L-arabinose concentrations (as indicated). Cells equivalent to one A600nm unit were collected from each culture and lysed in SDS-PAGE loading buffer. One half of each sample was treated with proteinase K, separated by SDS-PAGE and the LPS stained with Emerald Pro Q (Life Technologies). The remaining half was separated by SDS-PAGE and His6-WbdD was detected by Western blotting using anti-Penta-His antibody (Qiagen) together with horseradish peroxidase-conjugated goat-anti mouse antibody (Cedar Lane) and Luminata Classico Western HRP substrate (Millipore). Protein levels were quantitated by densitometry and are reported below the figure as the amounts relative to that observed in the sample with the highest expression level (0.05% L-arabinose).
