2012
DOI: 10.1007/978-1-61779-564-0_13
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Cloning Whole Bacterial Genomes in Yeast

Abstract: Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoni… Show more

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Cited by 31 publications
(39 citation statements)
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“…The central technique for building large heterologous DNA constructs in yeast is transformationassociated recombination (TAR), which leverages yeast's unique capability to efficiently combine many DNA fragments by homologous recombination in a single step (Larionov et al, 1996). Recently, researchers have demonstrated the ability to assemble and clone whole bacterial genomes in yeast (Gibson et al, 2008) using a combination of enzymatic in vitro assembly (Gibson, 2011) and TAR-based assembly (Benders et al, 2010) methods. This foundational advance opens up the possibility of rapidly assembling large biosynthetic pathways using a combination of oligonucleotide synthesis, in vitro assembly, and yeast-mediated TAR assembly, providing a scalable alternative to traditional recombinant DNA cloning methods.…”
Section: Rapid Assembly Of Biosynthetic Pathwaysmentioning
confidence: 99%
“…The central technique for building large heterologous DNA constructs in yeast is transformationassociated recombination (TAR), which leverages yeast's unique capability to efficiently combine many DNA fragments by homologous recombination in a single step (Larionov et al, 1996). Recently, researchers have demonstrated the ability to assemble and clone whole bacterial genomes in yeast (Gibson et al, 2008) using a combination of enzymatic in vitro assembly (Gibson, 2011) and TAR-based assembly (Benders et al, 2010) methods. This foundational advance opens up the possibility of rapidly assembling large biosynthetic pathways using a combination of oligonucleotide synthesis, in vitro assembly, and yeast-mediated TAR assembly, providing a scalable alternative to traditional recombinant DNA cloning methods.…”
Section: Rapid Assembly Of Biosynthetic Pathwaysmentioning
confidence: 99%
“…While intensive efforts have been made to turn E. coli into a universal molecular biology tool, the power of S. cerevisiae as DNA assembly platform has so far been largely untapped. Despite the pivotal role played by S. cerevisiae in the last decade in the assembly of genomes (14,(43)(44)(45)(46) there is so far relatively little effort invested in turning this yeast into a universal and powerful DNA assembly platform (47,48), a situation that we expect to see change in the future.…”
Section: Discussionmentioning
confidence: 99%
“…Yeast and Mycoplasma strains used in this study and culture conditions Saccharomyces cerevisiae strain W303a containing the marked Mmc genome YCpMmyc1.1 (Lartigue et al, 2009;Benders et al, 2010) and its derivative YCpMmyc1.1-Δglf were grown on synthetic minimal medium containing glucose (SD) either lacking histidine, or histidine and uracil, or lacking histidine and supplemented with 1 mg ml −1 5-fluoroorotic acid (5-FOA, Carl Roth, Germany) (Kouprina and Larionov, 2008;Noskov et al, 2010).…”
Section: Methodsmentioning
confidence: 99%