Direct transfer of whole genomes from bacteria to yeast

Karas, Bogumil J.; Jablanovic, Jelena; Sun, Lijie; Ma, Li; Goldgof, Gregory M.; Stam, Jason; Ramon, Adi; Manary, Micah J; Winzeler, Elizabeth A.; Venter, J. Craig; Weyman, Philip D.; Gibson, Daniel G.; Glass, John I.; Hutchison, Clyde A.; Smith, Hamilton O.; Suzuki, Yo

doi:10.1038/nmeth.2433

Cited by 68 publications

(118 citation statements)

References 23 publications

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“…It all began with cloning experiments and transposing the entire M. mycoïdes genome into a yeast cell (from which the original DNA was removed) (Benders et al 2010). Keeping this cloned genome in a plasmid has also been done (Karas et al 2013), and later transposing it again into the cell membrane of another mycoplasma species (M. capricolum) (Lartigue et al 2009). This mycoplasma to yeast genome transfection technology has also made possible new methods of reducing the genome, in the continuous search for the minimal essential gene set.…”

Section: Construction Of An Artificial Cellmentioning

confidence: 99%

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Molecular biology of mycoplasmas: from the minimum cell concept to the artificial cell

Córdova¹,

Hoeltgebaum

Machado

et al. 2016

An. Acad. Bras. Ciênc.

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Mycoplasmas are a large group of bacteria, sorted into different genera in the Mollicutes class, whose main characteristic in common, besides the small genome, is the absence of cell wall. They are considered cellular and molecular biology study models. We present an updated review of the molecular biology of these model microorganisms and the development of replicative vectors for the transformation of mycoplasmas. Synthetic biology studies inspired by these pioneering works became possible and won the attention of the mainstream media. For the fi rst time, an artifi cial genome was synthesized (a minimal genome produced from consensus sequences obtained from mycoplasmas). For the fi rst time, a functional artifi cial cell has been constructed by introducing a genome completely synthesized within a cell envelope of a mycoplasma obtained by transformation techniques. Therefore, this article offers an updated insight to the state of the art of these peculiar organisms' molecular biology.

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Section: Construction Of An Artificial Cellmentioning

confidence: 99%

“…The cloning of genomes in yeast, in addition to having been recreated with other bacteria, was also demonstrated with eukaryotic chromosomes (Karas et al 2013).…”

Section: Construction Of An Artificial Cellmentioning

confidence: 99%

Molecular biology of mycoplasmas: from the minimum cell concept to the artificial cell

Córdova¹,

Hoeltgebaum

Machado

et al. 2016

An. Acad. Bras. Ciênc.

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“…The transplantation was further improved by direct transfer of whole genomes from bacteria to yeast by fusion of bacterial cells with yeast spheroplasts. [79] Synthetic yeast 2.0…”

Section: Introductionmentioning

confidence: 99%

High molecular weight DNA assembly in vivo for synthetic biology applications

Juhas

Ajioka

2016

Critical Reviews in Biotechnology

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DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

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“…Because we can (1) introduce this genome into yeast and maintain it as a plasmid Karas et al 2013a); and (2) ''transplant'' the genome from yeast into mycoplasma recipient cells (Lartigue et al 2009), genetic tools in yeast are available for reducing this bacterial genome. Several systems offer advanced tools for bacterial genome engineering.…”

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confidence: 99%

“…One effective approach is to reuse the same marker after precise and scarless marker excision (Storici et al 2001). We have previously used a self-excising marker ) six times in yeast to generate a JCVI-syn1.0 genome lacking all six restriction systems (JCVI-syn1.0 Δ1-6) (Karas et al 2013a). Despite the advantages of scarless engineering, sequential procedures are time-consuming.…”

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confidence: 99%

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

et al. 2015

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The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and~10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.

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Direct transfer of whole genomes from bacteria to yeast

Cited by 68 publications

References 23 publications

Molecular biology of mycoplasmas: from the minimum cell concept to the artificial cell

Molecular biology of mycoplasmas: from the minimum cell concept to the artificial cell

High molecular weight DNA assembly in vivo for synthetic biology applications

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

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