Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Abstract

Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.

Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome.

Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains.

Figure 1

The pDOC donor plasmids. Circular representation of the pEX100T plasmid showing the location of the origins of replication, the sacB gene and the ampicillin resistance gene. Below is a linear representation of the pDOC plasmid inserts, showing the I-SceI restriction sites, cloning regions (CR, CR1 and CR2), the Flp recognition sites flanking the kanamycin resistance cassette (Kan^R) and the location of the epitope tags in plasmids pDOC-H, pDOC-F, pDOC-P and pDOC-G.

Figure 2

DNA sequences of the pDOC plasmids. (A) DNA sequence of pDOC-C insert. The location of sequencing primer annealing sites is indicated (SS1 and SS2). The I-SceI recognition sites are shown flanking the cloning region. (B) DNA sequences of the pDOC-K, pDOC-H, pDOC-F, pDOC-P and pDOC-G inserts. Sequences specific to each plasmid are shown in the open box. The first codon of the epitope tags are highlighted in grey, and the stop codons are indicated. The following primer annealing sites are indicated: SS1 and SS2, used to sequence plasmid derivatives pre-recombination; K-FWD, used for amplifying PCR products from pDOC-K for generating gene deletions; CC1 and CC2, used for generating PCR products in order to confirm recombination; P-REV, used to generate PCR products for cloning into pDOC-C pre-recombination. The Flp recognition sequences are shown (Flp1 and Flp2), flanking the kanamycin cassette. The cloning regions, CR1 and CR2 are shown, adjacent to the I-SceI recognition sites.

Figure 3

Schematic of pDOC based recombination. PCR products are generated for gene coupling (A) or for gene deleting (B) and cloned into pDOC-C. Homologous regions (H1-4) on the PCR product recombine with the target gene on the chromosome. Recombinant clones are then checked by PCR using primers annealing to the CC1 and CC2 sequences, and sequences adjacent to the homology regions.

Figure 4

G-DOC recombineering. The pDOC donor plasmid and the recombineering plasmid pACBSCE are co-transformed into the recipient strain. Arabinose induction promotes expression of the λ-Red gene products and I-SceI. I-SceI generates a linear DNA fragment form the donor plasmid that is a substrate for recombination with the chromosome mediated by the λ-Red system. Recombinants are selected by the ability to survive and grow on LB supplemented with kanamycin and sucrose.

Figure 5

Verification and functionality of chromosomal lacI::tag fusions. (A) Ethidum bromide stained agarose gel showing DNA amplified by PCR from the lacI fusion strains. Lanes 1 and 6 are DNA markers, lanes 2, 3, 4 + 5 show DNA derived from lacI::6 × his, lacI::3 × FLAG, lacI::ProteinA and lacI::GFP respectively. (B) Western blot analysis of tagged strains. Lanes 1, 4 and 7 show protein standards. Lanes 2, 5 and 8 show wild-type MG1655. Lanes 3, 6 and 9 show the tagged strains. Lanes 1, 2 + 3 were probed with antibody specific to the FLAG tag, lanes 4, 5 +6 were probed with antibody specific to ProteinA and lanes 7, 8 + 9 were probed with antibody specific to GFP. (C) SDS-PAGE analysis of the affinity-isolation of LacI::6 × His. Proteins were stained with Coomassie blue. Lane 1 shows protein standards, lane 2, whole cell extract, lane 3, LacI::6 × His affinity-isolate.