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dc.contributor.authorMosberg, Joshua Adam Weintrob
dc.contributor.authorGregg, Christopher
dc.contributor.authorLajoie, Marc Joseph
dc.contributor.authorWang, Harris He
dc.contributor.authorChurch, George McDonald
dc.date.accessioned2013-04-15T16:50:16Z
dc.date.issued2012
dc.identifier.citationMosberg, Joshua A., Christopher J. Gregg, Marc J. Lajoie, Harris H. Wang, and George M. Church. 2012. Improving Lambda Red genome engineering in Escherichia coli via rational removal of endogenous nucleases. PLoS ONE 7(9): e44638.en_US
dc.identifier.issn1932-6203en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10533604
dc.description.abstractLambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3′ ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.en_US
dc.language.isoen_USen_US
dc.publisherPublic Library of Scienceen_US
dc.relation.isversionofdoi:10.1371/journal.pone.0044638en_US
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434165/pdf/en_US
dash.licenseLAA
dc.subjectBiologyen_US
dc.subjectBiochemistryen_US
dc.subjectEnzymesen_US
dc.subjectEnzyme Classesen_US
dc.subjectNucleaseen_US
dc.subjectNucleic Acidsen_US
dc.subjectDNAen_US
dc.subjectDNA recombinationen_US
dc.subjectBiotechnologyen_US
dc.subjectGeneticsen_US
dc.subjectGenetic Mutationen_US
dc.subjectMutagenesisen_US
dc.subjectModel Organismsen_US
dc.subjectProkaryotic Modelsen_US
dc.subjectEscherichia Colien_US
dc.subjectMolecular Cell Biologyen_US
dc.subjectSynthetic Biologyen_US
dc.titleImproving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleasesen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalPLoS ONEen_US
dash.depositing.authorChurch, George McDonald
dc.date.available2013-04-15T16:50:16Z
dc.identifier.doi10.1371/journal.pone.0044638*
dash.contributor.affiliatedMosberg, Joshua Adam Weintrob
dash.contributor.affiliatedGregg, Christopher
dash.contributor.affiliatedLajoie, Marc
dash.contributor.affiliatedWang, Harris He
dash.contributor.affiliatedChurch, George


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