Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains
<p>Abstract</p> <p>Background</p> <p>Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in <it>Escherichia coli</it>. Several protocols have been developed that differ in the mechanisms by which DNA,...
| Main Authors: | , , , , , , |
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| Format: | Article |
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BMC
2009-12-01
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| Series: | BMC Microbiology |
| Online Access: | http://www.biomedcentral.com/1471-2180/9/252 |
| _version_ | 1818823488285179904 |
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| author | Penn Charles W Pallen Mark J Heurlier Karin Bingle Lewis EH Lee David J Busby Stephen JW Hobman Jon L |
| author_facet | Penn Charles W Pallen Mark J Heurlier Karin Bingle Lewis EH Lee David J Busby Stephen JW Hobman Jon L |
| author_sort | Penn Charles W |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in <it>Escherichia coli</it>. 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 <it>in vivo </it>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 <it>E. coli </it>K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic <it>E. coli </it>strains, which has led us to develop an enhanced protocol for recombineering in such strains.</p> <p>Results</p> <p>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 <it>E. coli </it>strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved <it>in vivo </it>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 <it>sacB </it>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.</p> <p>Conclusion</p> <p>We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in <it>E. coli</it>. 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) <it>E. coli </it>strains as well as in K-12 laboratory strains.</p> |
| first_indexed | 2024-12-18T23:40:46Z |
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| last_indexed | 2024-12-18T23:40:46Z |
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| spelling | doaj.art-974eead8b8d64b09980336fb2ffe226c2022-12-21T20:47:24ZengBMCBMC Microbiology1471-21802009-12-019125210.1186/1471-2180-9-252Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strainsPenn Charles WPallen Mark JHeurlier KarinBingle Lewis EHLee David JBusby Stephen JWHobman Jon L<p>Abstract</p> <p>Background</p> <p>Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in <it>Escherichia coli</it>. 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 <it>in vivo </it>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 <it>E. coli </it>K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic <it>E. coli </it>strains, which has led us to develop an enhanced protocol for recombineering in such strains.</p> <p>Results</p> <p>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 <it>E. coli </it>strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved <it>in vivo </it>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 <it>sacB </it>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.</p> <p>Conclusion</p> <p>We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in <it>E. coli</it>. 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) <it>E. coli </it>strains as well as in K-12 laboratory strains.</p>http://www.biomedcentral.com/1471-2180/9/252 |
| spellingShingle | Penn Charles W Pallen Mark J Heurlier Karin Bingle Lewis EH Lee David J Busby Stephen JW Hobman Jon L Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains BMC Microbiology |
| title | Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains |
| title_full | Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains |
| title_fullStr | Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains |
| title_full_unstemmed | Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains |
| title_short | Gene doctoring: a method for recombineering in laboratory and pathogenic <it>Escherichia coli </it>strains |
| title_sort | gene doctoring a method for recombineering in laboratory and pathogenic it escherichia coli it strains |
| url | http://www.biomedcentral.com/1471-2180/9/252 |
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