Journal: Molecular Microbiology

Article Title: Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi

doi: 10.1111/j.1365-2958.2007.05969.x

Figure Lengend Snippet: A. PCR analysis of genomic DNA from B. burgdorferi clones transformed with gene inactivation constructs targeting genes in the bbb26–27 region. Template DNAs from transformants are identified below the lanes and PCR amplification targets above the lanes. Template DNA from B31-A illustrates the PCR products from the wild-type alleles of bbb26–27 , bbb26 and bbb27 (lanes 2–5, 15 and 19), whereas template DNAs from the gene inactivation constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ) depict the PCR profiles of the mutated alleles (lanes 6–9, 16 and 20). The PCR products resulting from the clones transformed with the allelic exchange inactivation plasmids are illustrated in lanes 10–13, 17 and 21. The 1 kbp-plus size standards (Invitrogen) were run in lanes 1, 14 and 18 and sizes (base pairs) are indicated to the left of the panel. B. Graphical representation of the bbb26–27 region on cp26 (B31-A) and the cloned pieces of DNA used for the allelic exchange constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ). The 1168 bp region of cp26 between nucleotides 21923 and 23091 was replaced with the 1146 bp flaB p – aadA resistance cassette to create XL-BBB26–27Δ for disruption of both bbb26 and bbb27 . The 742 bp region of cp26 between nucleotides 21923 and 22579 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB26Δ for disruption of bbb26 . The 438 bp region of cp26 between nucleotides 22653 and 23091 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB27Δ for disruption of bbb27 . Locations of the primers used for analysis in (A) are indicated and the sequences are listed in .

Article Snippet: Total genomic DNA of B. burgdorferi was isolated from 15 ml of cultures with a Wizard genomic DNA purification kit (Promega).

Techniques: Clone Assay, Transformation Assay, Construct, Amplification, Disruption

Journal: Molecular Microbiology

Article Title: Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi

doi: 10.1111/j.1365-2958.2007.05969.x

Figure Lengend Snippet: 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; flgB P – 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.

Article Snippet: Total genomic DNA of B. burgdorferi was isolated from 15 ml of cultures with a Wizard genomic DNA purification kit (Promega).

Techniques: Plasmid Preparation, Marker, Southern Blot, Transformation Assay