Multicopy plasmid modification with phage λ Red recombineering

Abstract

Recombineering, in vivo genetic engineering using the bacteriophage λ Red generalized recombination system, was used to create various modifications of a multicopy plasmid derived from pBR322. All genetic modifications possible on the E. coli chromosome and on bacterial artificial chromosomes (BACs) are also possible on multicopy plasmids and are obtained with similar frequencies to their chromosomal counterparts, including creation of point mutations (5-10% unselected frequency), deletions and substitutions. Parental and recombinant plasmids are nearly always present as a mixture following recombination, and circular multimeric plasmid molecules are often generated during the recombineering. Keywordsphage λ Red homologous recombination; multicopy plasmid; plasmid multimers; in vivo genetic engineering

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Counterselectable markers can also be used to recycle selectable markers and to generate strains with multiple gene knockouts, which is desirable due to the limited number of selectable markers available for use in Chlamydia. In addition, knockout strains might eventually be constructed by recombineering using bacteriophage Red recombinase (165). With this system, targeted gene disruption can be achieved in vivo by cotransforming a linear donor sequence together with a suicide plasmid expressing the Red recombinase.…”

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

“…With this system, single-stranded DNA (ssDNA) oligonucleotides have been used to efficiently modify E. coli chromosomal targets Costantino and Court 2003), BACs (Swaminathan et al 2001), and plasmids (Thomason et al 2007b), as well as to rapidly optimize a metabolic pathway coding for the production of lycopene (Wang et al 2009). Furthermore, linear double-stranded DNA (dsDNA) recombineering has been used to replace chromosomal genes (Murphy 1998;Murphy et al 2000), to disrupt gene function (Datsenko and Wanner 2000), and to develop novel cloning methods Li and Elledge 2005).…”

Efficient Generation of Unmarked Deletions in Legionella pneumophila

“…To facilitate construction of strains with multiple unmarked nonpolar deletions, we coupled phage-mediated recombination in Escherichia coli with Flp-mediated excision in L. pneumophila. By exploiting a phage enzyme to mediate homologous recombination between DNA substrates with as few as 35 nucleotides of homology, so-called recombineering offers several advantages over restriction enzymebased cloning, including increased efficiency (8,10,27,28,31). The Saccharomyces cerevisiae Flp site-specific recombinase excises DNA flanked by directly repeated 34-bp FRT sites (9,12,13,19,21,23,24).…”

“…The gene of interest was replaced by an FRT-flanked cat or kan cassette from pKD3 or pKD4, respectively (10), using recombineering and E. coli DY330 (Table 1) (27,28). The recombinant allele from pGEM was then transferred to the Lp02 chromosome by natural transformation (25,26).…”