Differential requirements of singleplex and multiplex recombineering of large DNA constructs
Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains an...
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| description | Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency. |
| doi_str_mv | 10.1371/journal.pone.0125533 |
| format | Article |
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The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0125533</identifier><identifier>PMID: 25954970</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Antibiotics ; Artificial chromosomes ; Bacteria ; Bacteriophage lambda - genetics ; Biochemistry ; Cassettes ; Catalysis ; Cloning, Molecular ; Construction ; Deoxyribonucleic acid ; Design engineering ; Design parameters ; DNA ; DNA - genetics ; E coli ; Efficiency ; Enzymes ; Escherichia coli ; Genetic engineering ; Genetic Engineering - methods ; Genetically modified organisms ; Genomes ; Genomics ; Homologous recombination ; Homologous Recombination - genetics ; Homology ; In vivo methods and tests ; Insertion ; Metabolism ; Metabolites ; Multiplexing ; Mutation ; Phages ; Phosphorothioate ; Plasmids ; Proteins</subject><ispartof>PloS one, 2015-05, Vol.10 (5), p.e0125533-e0125533</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Reddy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Reddy et al 2015 Reddy et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c641t-e447d0dfcc997c9ec6c7aec598cfb7305f781742b173c2b882a5fc1b3803ca213</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/PMC4425527/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425527/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25954970$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Reddy, Thimma R</creatorcontrib><creatorcontrib>Kelsall, Emma J</creatorcontrib><creatorcontrib>Fevat, Léna M S</creatorcontrib><creatorcontrib>Munson, Sarah E</creatorcontrib><creatorcontrib>Cowley, Shaun M</creatorcontrib><title>Differential requirements of singleplex and multiplex recombineering of large DNA constructs</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Reddy, Thimma R</au><au>Kelsall, Emma J</au><au>Fevat, Léna M S</au><au>Munson, Sarah E</au><au>Cowley, Shaun M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Differential requirements of singleplex and multiplex recombineering of large DNA constructs</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-05-08</date><risdate>2015</risdate><volume>10</volume><issue>5</issue><spage>e0125533</spage><epage>e0125533</epage><pages>e0125533-e0125533</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25954970</pmid><doi>10.1371/journal.pone.0125533</doi><oa>free_for_read</oa></addata></record> |
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| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Antibiotics Artificial chromosomes Bacteria Bacteriophage lambda - genetics Biochemistry Cassettes Catalysis Cloning, Molecular Construction Deoxyribonucleic acid Design engineering Design parameters DNA DNA - genetics E coli Efficiency Enzymes Escherichia coli Genetic engineering Genetic Engineering - methods Genetically modified organisms Genomes Genomics Homologous recombination Homologous Recombination - genetics Homology In vivo methods and tests Insertion Metabolism Metabolites Multiplexing Mutation Phages Phosphorothioate Plasmids Proteins |
| title | Differential requirements of singleplex and multiplex recombineering of large DNA constructs |
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