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Figure 1 | Veterinary Research

Figure 1

From: Safety assessment of compliant, highly invasive, lipid A-altered, O-antigen-defected Salmonella strains as prospective vaccine delivery systems

Figure 1

Diagrammatic representation for deletion and substitution of genes using the λ red recombinase system. A. Deletion of the target gene (lon/cpxR/sifA/rfaL/asd). catR was amplified from the pKD3 plasmid with the aid of primers designed by incorporating homologous regions from the target gene to be deleted. Bacterial strains were transformed with pKD46 to express λ red recombinase. Then, the amplified gene was transformed into pKD46 transformants, and the chloramphenicol-resistant transformants assured the deletion of the target gene. B. Substitution of lpxE for pagL. lpxE and catR were amplified and ligated. The ligated construct was amplified using primers designed based on upstream and downstream homologous regions of pagL. pKD46 transformant bacterial strains were electroporated with the amplified linear DNA. Screening in chloramphenicol-containing plates confirmed the replacement of pagL. catR was eliminated with the expression of pCP20. H1 and H2—homologous regions, FP, RP—forward and reverse primers, FRT-1/2—flippase recognition target, catR—chloramphenicol acyl transferase.

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