Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

Abstract

BackgroundEscherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.ResultsThe PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.ConclusionsBy retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones

“…As a subset of the plasmids were to contain the RT-cassette which, we had found, confers instability when present in a standard high-copy number vector (unpublished observations), it was decided to base them all in pCC1Fos. As well as maintaining clones as single copies for stable, routine propagation pCC1Fos-based constructs can, when hosted in a suitable strain such as EPI300 or MW005 [6], also be transiently induced to approximately 50 copies per bacterial cell thus improving DNA isolation yield and purity. These plasmids were designed as pairs the first member of which provides the template for PCR-amplification of the RT-cassette, used in the first, positive selection recombineering step, while the second provides the desired replacement sequence, excised as a restriction fragment, for use in the subsequent counter selection step.…”

“…To facilitate the generation of such reporter constructs from C. elegans fosmid clones we [5,6], and others [7-9], have developed tools and techniques designed to leverage the power of recombineering. Recombineering is a homologous recombination (HR)-based genetic engineering system mediated by transient expression of λ -encoded recombinases within an E .…”

Identification of Spodoptera exigua nucleopolyhedrovirus genes involved in pathogenicity and virulence

“…PCR on pCRTopo-lox-cat-lox was performed using Phusion Polymerase (Thermo Scientific) according to the manufacturer, giving a product of 1170 bp. SeBacAL1 DNA was cloned into electrocompetent MW003 cells (Westenberg et al, 2010), and selected on LB-plates with streptomycin and kanamycin for 2 d at 32°C. Single colonies were picked and used to inoculate 1 ml SOB-medium at 32°C.…”

λ Recombination and Recombineering

“…Dolphin and Hope used recombineering to construct a marker-free gfp translational fusion in a Caenorhabditis elegans fosmid clone, which was subsequently used to generate transgenic worms for expression analysis (287). More recent descriptions of fosmid recombineering by this group describes the use of a strain that can induce higher copy numbers of recombineered BACs and fosmids following recombineering (288), and the use of complementary sets of constructs for the two-step generation of genetic fusions in C. elegans genomic clones (289).…”