Fig 3. Generation of suicide plasmid for mutagenesis in which B. burgdorferi-codon optimized firefly luciferase gene and streptomycin resistance cassette in tandem disrupt bbe02 gene.

The bbe02 gene was amplified from the B31 genome using primers BBE02Nde-long and BBE02Xho-long, and amplicon was cloned into pCR-XL-TOPO vector to form pXbbe02. The plasmid pXbbe02 digested with NheI and PacI restriction enzymes liberated the internal fragment of the bbe02 gene. B. burgdorferi-codon optimized firefly luciferase gene under the flaB promoter (PflaB-Bbluc) amplified from plasmid pJSB175 was then cloned using XbaI and PacI restriction enzymes, in the compatible NheI and PacI overhangs of the pXbbe02 to form pXbbe02Bbluc. The streptomycin resistance cassette was then cloned into the PacI site at the 3’ end of the Bbluc gene, to form pXbbe02Bbluc-aadA. This plasmid was then used to transform infectious B. burgdorferi to generate bioluminescent N40 strain with disrupted bbe02 gene.