Fig. 2: Western blot and spot blot analysis investigating minimal gene cluster requirements for recombinant RhaPS production in E. coli.

A E. coli rfaS deletion cells transformed with two plasmids containing either the Shi1/2a gene cluster fragment, GacB control or empty plasmid (negative control) as well as the RhaPS clusters (ΔB) were grown overnight and whole cell lysates subjected to SDS-PAGE and western blotting with the commercially available anti-GAC antibodies. Plasmids indicated: ΔB = gacACDEFG; B = gacB; Shi1 = wbbPQR, Shi2a = rfbFG, and empty plasmid controls=-. B E. coli rfaS deletion cells transformed with combinations of RhaPS cluster, and Shi1 gene cluster fragments were grown overnight and subjected to dot blot analysis using the GAC antibodies. RhaPS is synthesised by the WbbPQR proteins when co-expressed with the ΔB RhaPS proteins. Omitting one of the Shi1 proteins results in weaker or no RhaPS production.