Manipulating Replisome Dynamics to Enhance Lambda Red-Mediated Multiplex Genome Engineering

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Manipulating Replisome Dynamics to Enhance Lambda Red-Mediated Multiplex Genome Engineering

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Title: Manipulating Replisome Dynamics to Enhance Lambda Red-Mediated Multiplex Genome Engineering
Author: Lajoie, Marc Joseph; Gregg, Christopher; Mosberg, Joshua Adam Weintrob; Washington, G. C.; Church, George McDonald

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Citation: Lajoie, Marc Joseph, Christopher J. Gregg, Joshua Adam Weintrob Mosberg, G. C. Washington, and George McDonald Church. 2012. Manipulating replisome dynamics to enhance lambda red-mediated multiplex genome engineering. Nucleic Acids Research 40(22): e170.
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Abstract: Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.
Published Version: doi:10.1093/nar/gks751
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526312/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:11177944
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