Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes
A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence f...
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| Veröffentlicht in: | Applied and Environmental Microbiology 2006-04, Vol.72 (4), p.2520-2525 |
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| container_title | Applied and Environmental Microbiology |
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| creator | Bloor, Alexandra E Cranenburgh, Rocky M |
| description | A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary. |
| doi_str_mv | 10.1128/AEM.72.4.2520-2525.2006 |
| format | Article |
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This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>DOI: 10.1128/AEM.72.4.2520-2525.2006</identifier><identifier>PMID: 16597952</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>Bacillus subtilis ; Bacillus subtilis - genetics ; Bacillus subtilis - growth & development ; Bacteria ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biological and medical sciences ; Biotechnology ; Chromosomes, Bacterial - genetics ; Drug Resistance, Bacterial - genetics ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - growth & development ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - metabolism ; Fundamental and applied biological sciences. Psychology ; Gene Deletion ; Genes, Bacterial ; Genetic engineering ; Genetic Markers ; Genetic recombination ; Genetic Techniques ; Genetics and Molecular Biology ; Integrases - genetics ; Integrases - metabolism ; Microbiology ; Recombination, Genetic</subject><ispartof>Applied and Environmental Microbiology, 2006-04, Vol.72 (4), p.2520-2525</ispartof><rights>2006 INIST-CNRS</rights><rights>Copyright American Society for Microbiology Apr 2006</rights><rights>Copyright © 2006, American Society for Microbiology 2006</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c607t-5548237f867a51d9eca47bf02114230561f720e21eaf04ccab8450fbc86e95c3</citedby><cites>FETCH-LOGICAL-c607t-5548237f867a51d9eca47bf02114230561f720e21eaf04ccab8450fbc86e95c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449051/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449051/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,3186,3187,27922,27923,53789,53791</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=17700610$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16597952$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bloor, Alexandra E</creatorcontrib><creatorcontrib>Cranenburgh, Rocky M</creatorcontrib><title>Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes</title><title>Applied and Environmental Microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.</description><subject>Bacillus subtilis</subject><subject>Bacillus subtilis - genetics</subject><subject>Bacillus subtilis - growth & development</subject><subject>Bacteria</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chromosomes, Bacterial - genetics</subject><subject>Drug Resistance, Bacterial - genetics</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - growth & development</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Deletion</subject><subject>Genes, Bacterial</subject><subject>Genetic engineering</subject><subject>Genetic Markers</subject><subject>Genetic recombination</subject><subject>Genetic Techniques</subject><subject>Genetics and Molecular Biology</subject><subject>Integrases - genetics</subject><subject>Integrases - metabolism</subject><subject>Microbiology</subject><subject>Recombination, Genetic</subject><issn>0099-2240</issn><issn>1098-5336</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkt9v0zAQxyMEYmXwL7CABG8pZ8c_kpdJoyoDaRXSNiTeLMc9tx5JXOx0sP8eR40o8MLLWTp_7ntn3zfLzgjMCaHVu4vlai7pnM0pp1CkwOcUQDzKZgTqquBlKR5nM4C6LihlcJI9i_EOABiI6ml2QgSvZc3pLLtfWuuMw37IVzhs_Tr3Nr_BFs2gmxbzlQ7fMOSX2GO-_GlcdL7Pm4f8a0peo_Fd43o9jEnrJ-wad6022I2ars_fazNgcLrNF9vgOx99h_F59sTqNuKL6TzNbj8sbxcfi6vPl58WF1eFESCHgnNW0VLaSkjNybpGo5lsLFBCGC2BC2IlBaQEtQVmjG4qxsE2phJYc1OeZucH2d2-6XBt0khBt2oXXKfDg_Laqb9verdVG3-vCGM1cJIE3k4CwX_fYxxU56LBttU9-n1UQlYlk-T_IAXKhJQiga__Ae_8PvTpExKTtsJ4CQmSB8gEH2NA-3tkAmo0gEoGUJIqpkYDjIGr0QCp8uWfLz7WTRtPwJsJ0NHo1gbdp60eOSmTDBlHeHXgtm6z_eECKh07pbE7tk3M2YGx2iu9CUnnyw0FUgIBIVnFy18sg84X</recordid><startdate>20060401</startdate><enddate>20060401</enddate><creator>Bloor, Alexandra E</creator><creator>Cranenburgh, Rocky M</creator><general>American Society for Microbiology</general><scope>FBQ</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7SN</scope><scope>7SS</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>SOI</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20060401</creationdate><title>Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes</title><author>Bloor, Alexandra E ; Cranenburgh, Rocky M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c607t-5548237f867a51d9eca47bf02114230561f720e21eaf04ccab8450fbc86e95c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Bacillus subtilis</topic><topic>Bacillus subtilis - genetics</topic><topic>Bacillus subtilis - growth & development</topic><topic>Bacteria</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Chromosomes, Bacterial - genetics</topic><topic>Drug Resistance, Bacterial - genetics</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - growth & development</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Deletion</topic><topic>Genes, Bacterial</topic><topic>Genetic engineering</topic><topic>Genetic Markers</topic><topic>Genetic recombination</topic><topic>Genetic Techniques</topic><topic>Genetics and Molecular Biology</topic><topic>Integrases - genetics</topic><topic>Integrases - metabolism</topic><topic>Microbiology</topic><topic>Recombination, Genetic</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bloor, Alexandra E</creatorcontrib><creatorcontrib>Cranenburgh, Rocky M</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Applied and Environmental Microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bloor, Alexandra E</au><au>Cranenburgh, Rocky M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes</atitle><jtitle>Applied and Environmental Microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>2006-04-01</date><risdate>2006</risdate><volume>72</volume><issue>4</issue><spage>2520</spage><epage>2525</epage><pages>2520-2525</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><coden>AEMIDF</coden><abstract>A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.</abstract><cop>Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>16597952</pmid><doi>10.1128/AEM.72.4.2520-2525.2006</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacillus subtilis Bacillus subtilis - genetics Bacillus subtilis - growth & development Bacteria Bacterial Proteins - genetics Bacterial Proteins - metabolism Biological and medical sciences Biotechnology Chromosomes, Bacterial - genetics Drug Resistance, Bacterial - genetics Enzymes Escherichia coli Escherichia coli - genetics Escherichia coli - growth & development Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Fundamental and applied biological sciences. Psychology Gene Deletion Genes, Bacterial Genetic engineering Genetic Markers Genetic recombination Genetic Techniques Genetics and Molecular Biology Integrases - genetics Integrases - metabolism Microbiology Recombination, Genetic |
| title | Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes |
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