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

Abstract

BackgroundHomologous recombination mediated by the λ-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 λ-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 λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.ResultsOur 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 λ-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 6 × His, 3 × FLAG, 4 × ProteinA 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 λ-Red system, which can lead to unwanted secondary alterations to the chromosome.ConclusionWe 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.

Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

“…The ⌬surA ⌬fkpA and ⌬skp ⌬fkpA mutant strains were constructed from the ⌬surA and ⌬skp single mutant strains, respectively, by using the gene-doctoring system (34). For this purpose, we constructed two plasmids, pACE and pDOC-fkpA-Tc.…”

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

“…Briefly, the BamA-K135Bpa strain was constructed as follows. First, we modified the wild-type strain BW25113 using a gene doctoring strategy as described earlier (34) to generate the strain LY928, in which the genome encoded the orthogonal aminoacyl-tRNA synthetase and tRNA required for Bpa incorporation. Via the same strategy, the genome of the LY928 strain was further modified to replace the wild type bamA gene with a construct encoding BamA-K135Bpa.…”

Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF

“…The first PCR fragment was obtained by amplification of 389 bp from the 39 end of the lapA gene without a stop codon using primers PP0168-10 and PP0168-2 (Table S1). The second PCR fragment was obtained by amplification of gfp and Km r genes from pDOC-G (Lee et al, 2009) using oligonucleotides Emgfp-5 and Km6. The third PCR fragment was obtained by amplification of the 352 bp DNA sequence downstream from the lapA gene using primers PP0168-3 and PP0168-40.…”

“…1) was employed for the generation of LapA-GFP fusion protein by inserting the gfp gene downstream of lapA in the P. putida chromosome. pDOC-lapA-gfp was constructed by using one-step ligation-independent cloning (SLIC) (Jeong et al, 2012) to insert three PCR fragments into pDOC-C (Lee et al, 2009) opened with EcoRI. The first PCR fragment was obtained by amplification of 389 bp from the 39 end of the lapA gene without a stop codon using primers PP0168-10 and PP0168-2 (Table S1).…”