Journal: Biotechnology Reports
Article Title: Genome modifications and cloning using a conjugally transferable recombineering system
Figure Lengend Snippet: Strategy for PCR-free cloning of large bacterial genetic regions. The major steps of cloning large genetic inserts are indicated. The catR-oriT-oriR (pMJH97) cassette was PCR amplified using primer pairs with 50–60 bp homologous sequence at their 5′-ends specific to the targetred site. Depending on the choice of restriction enzymes, the resulting dsDNA substrate can be integrated upstream or downstream of the targeted site of the genome using the recombineering system. Once the catR-oriT-oriR (pMJH97) cassette integration into the genome was confirmed by PCR and sequencing using primers P1 and P2, the genomic DNA of integrants was restriction digested with an appropriate restriction enzyme to clone into E. coli after self-ligation using T4 DNA ligase. The cloning of the correct insert into the plasmid pMJH97 was verified by PCR and sequencing using vector and insert specific primers P3 and P4, respectively. The plasmids with cloned inserts were then readily transfered to other Gram-negative bacterial strain by oriT sequence-mediated conjugal transfer using an appropriate donor strain.
Article Snippet: Blunt-ended and purified genomic DNA fragments were self-ligated using T4 DNA ligase and electroporated into E. coli (E. cloni 10G, Lucigen Corp.) for cloning.
Techniques: Polymerase Chain Reaction, Clone Assay, Amplification, Sequencing, Ligation, Plasmid Preparation