Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
Linear BAC Recombineering E. coliGenomic DNA Chromosome Phage N15 Plasmid
This is a preview of subscription content, log in to check access
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
The authors are grateful to Nikolai Ravin for providing N15 reagents and to Sek-Chuen Chow for support and encouragement. Q.C. is grateful to Monash University Malaysia for a HDR Scholarship. This work was partly funded by a Fundamental Research Grant Scheme FRGS/1/2011/ST/MUSM/02/2 from the Ministry of Higher Education Malaysia to K.N.
Kakeda M, Nagata K, Osawa K et al (2011) A new chromosome 14-based human artificial chromosome (HAC) vector system for efficient transgene expression in human primary cells. Biochem Biophys Res Commun 415:439–444PubMedCrossRefGoogle Scholar
Allardet-Servent A, Michaux-Charachon S, Jumas-Bilak E et al (1993) Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. J Bacteriol 175:7869–7874PubMedPubMedCentralGoogle Scholar
Lezhava A, Mizukami T, Kajitani T et al (1995) Physical map of the linear chromosome of Streptomyces griseus. J Bacteriol 177:6492–6498PubMedPubMedCentralGoogle Scholar
Hertwig S (2007) Linear plasmids and prophages in gram-negative bacteria. In: Meinhardt F, Klassen R (eds) Microbial linear plasmids. Springer, BerlinGoogle Scholar
Deneke J, Ziegelin G, Lurz R et al (2000) The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity. Proc Natl Acad Sci U S A 97:7721–7726PubMedCrossRefPubMedCentralGoogle Scholar
Ravin NV, Strakhova TS, Kuprianov VV (2001) The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form. J Mol Biol 312:899–906PubMedCrossRefGoogle Scholar
Ooi YS, Warburton PE, Ravin NV et al (2008) Recombineering linear DNA that replicate stably in E. coli. Plasmid 59:63–71PubMedCrossRefGoogle Scholar
Narayanan K, Williamson R, Zhang Y et al (1999) Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther 6:442–447PubMedCrossRefGoogle Scholar
Kaufman RM, Pham CT, Ley TJ (1999) Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome. Blood 94:3178–3184PubMedGoogle Scholar
Guzman LM, Belin D, Carson MJ et al (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedPubMedCentralGoogle Scholar
Grant SG, Jessee J, Bloom FR et al (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87:4645–4649PubMedCrossRefPubMedCentralGoogle Scholar