Fig. 3.
A. Graphical representation of pRB3, a cp26-incompatible shuttle vector that carries the resT, bbb26 and bbb27 genes of cp26. The bbb25 and bbb28 genes on pRB3 are truncated, lacking 3′ ends. Relevant restriction sites are shown. colE1, E. coli plasmid origin of replication; ZEO, zeocin-resistance marker; flgBP–kan, kanamycin resistance cassette; bbb10–13, ORFs from cp26 that confer autonomous plasmid replication and incompatibility with cp26 (Byram et al., 2004). B. Displacement of cp26 from B. burgdorferi by pRB3 as demonstrated by Southern blot analysis of transformants. Wild-type B. burgdorferi DNA (wt Bb), E. coli plasmid DNA of shuttle vector carrying resT, bbb26 and bbb27 (pRB3) and total genomic DNA from B. burgdorferi transformed with pRB3 (Bb/pRB3). Southern blot probed with the kanamycin resistance gene (left panel), which is present on pRB3, or the same blot stripped and reprobed with the resT gene (right panel), which is present on both cp26 and pRB3. An arrow indicates the supercoiled form of endogenous cp26; brackets denote supercoiled and nicked forms of pRB3. The mobilities of size standards (kbp) are indicated to the left of the panels.