<i>In vivo</i> cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in <i>Salmonella enterica</i>

Abstract

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. enzymes, which vary from insert to insert even for a given vector plasmid, and there is no need to purify and ligate DNA fragments, which is required in conventional recombinant techniques. This method, which relies on PCR amplification for the preparation of entire insert fragments (Bubeck et al., 1993;Jacobus and Gross, 2015;Li et al., 2011;Oliner et al., 1993;Spiliotis, 2012;Thieme et al., 2011) or entire plasmid clones (Jacobus and Gross, 2015;Li et al., 2011;Spiliotis, 2012), co-transformation, and recombinases, such as RecE/RecT from Rac prophage (Zhang et al., 1998) or Redabd from bacteriophage lambda (Bubeck et al., 1993;Jacobus and Gross, 2015;Li et al., 2011;Oliner et al., 1993;Spiliotis, 2012;Thieme et al., 2011), also allows several fragments to be combined into a vector at a time, thus reducing the cloning processing time. However, due to the high dependence on PCR, which produces errors in vitro and tends to amplify smaller fragments at a higher yield, its use for DNA fragments larger than 10 kb or so is unreliable. To be sure that no mistakes are produced during the construction of a clone and to perform clean genetics, sequencing of the entire PCR-generated DNA insert from many candidate clones is crucial.One-step inactivation methods have become common for constructing chromosomal deletion mutants in Gramnegative bacteria such as Escherichia coli and S. enterica (Datsenko and Wanner, 2000). In this method, a small (1, 1.3, or 1.5 kb) antibiotic marker cassette is amplified to possess approximately 40 bp regions homologous to upstream and downstream regions of the target gene, respectively, at both ends of the resulting cassette by PCR. Then, to facilitate double crossover events at the target gene on the chromosome, this linear DNA fragment is transformed into bacterial cells expressing lambda Red recombinase. Because the cassette was designed to harbor two FRT sites at the both ends of the antibiotic marker, it can be removed In vivo cloning of large chromosomal segments into a BAC derivative by generalized transduction and recombineering in Salmonella enterica (Received March 5, 2016 Recombineering has been used to facilitate the development of in vivo cloning methods. However, the method relies heavily on PCR, which still generates a much higher error rate than DNA replication in vivo, even when amplifying large DNA inserts. Here, a precise technique is reported in Salmonella enterica that enables the cloning of up to at least 19 kb target chromosomal DNA segments that had been marked by FRTs, which were derived from two consecutive lambda Red-mediated recombination events. P22 phage was utilized to transduce the target DNA segments from donor strains to recipient strains harboring a derivative of bacterial artificial chromosome (BAC) containing a FRT and a plasmid expressing Flp recombinase. This method was successful in cloning...

Isolation and draft genome sequence of Enterobacter asburiae strain i6 amenable to genetic manipulation

“…The results showed that the ATCC 35953 strain could not be transformed by the relatively large plasmid DNAs pCF430 (10 kb) 33 and pAK1001 (8.4 kb) 34 , whereas i6 formed colonies, showing transformation efficiency at least four orders of magnitude higher than that of ATCC 35953 especially with pCF430 (Fig. 1 ).…”

Development of conjugation-mediated versatile site-specific single-copy luciferase fusion system

“…Recombineering techniques that rely on λ Red recombinase and the FLP/FRT system have been used to construct large, precise clones (Kato, 2016) and precise gene fusions (Ellermeier et al, 2002). Here I extend these techniques to create a versatile, sitespecific, conjugation-mediated single-copy luciferase fusion system.…”