Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast

Lartigue, Carole; Vashee, Sanjay; Algire, Mikkel A.; Chuang, Ronald Y.; Benders, Gwynedd A.; Ma, Li; Noskov, Vladimir N.; Denisova, Evgeniya A.; Gibson, Daniel G.; Assad-García, Nacyra; Alperovich, Nina; Thomas, David W.; Merryman, Chuck; Hutchison, Clyde A.; Smith, Hamilton O.; Venter, J. Craig; Glass, John I.

doi:10.1126/science.1173759

Cited by 275 publications

(349 citation statements)

References 12 publications

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“…Given that specific double-strand DNA breaks are known to promote repair by homologous recombination and have formed the basis of robust technologies to seamlessly manipulate genomes (27,28), we hypothesized that coupling DNA cleavage by a homing endonuclease to DNA assembly could provide the needed boost in efficiency. Here we develop a method that utilizes endonuclease-stimulated homologous recombination for DNA assembly and demonstrate that we can easily and efficiently build large libraries of biosynthetic pathways in vivo.…”

mentioning

confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

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The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae , and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10 4 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

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confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Here we cloned natural genomes by transferring them from bacteria to yeast in one piece (18). This contrasts with the approach of assembling multiple smaller fragments into a genome, which is a strategy that others have used in attempting to clone the entire Synechocystis (15) and E. coli (14) genomes.…”

Section: Discussionmentioning

confidence: 99%

“…We have taken three different approaches to the cloning of bacterial genomes in yeast (Figure 1). The first approach is to insert a yeast vector into the bacterial genome prior to transformation of yeast (18,39,40) ( Figure 1A). …”

Section: Strategies For Cloning Bacterial Genomes In Yeastmentioning

confidence: 99%

Cloning Whole Bacterial Genomes in Yeast

Benders

2012

Methods in Molecular Biology

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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. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.

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“…First, the M. mycoides genome was resequenced and computationally disassembled into 1078 overlapping cassettes, each 1080 BP long [1]. These cassettes were chemically synthesized and implanted in yeast, where the chromosome repair machinery of yeast was used to assemble these strands into a complete chromosome [9,52,53]. Next, the complete chromosome was injected into a Mycoplasma capricolum cell, effectively rewriting the operating system of that cell with the instruction set from the injected M. mycoides genome [1].With this proof of principle complete, efforts are now beginning on the design and synthesis of reduced versions of the M. mycoides genome.…”

Section: Rewriting the Operating System Of Life From The Bottom Upmentioning

confidence: 99%

Building the blueprint of life

Henry

Overbeek²,

Stevens

2010

Biotechnology Journal

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With recent breakthroughs in experimental microbiology making it possible to synthesize and implant an entire genome to create a living cell, the challenge of constructing a working blueprint for the first truly minimal synthetic organism is more important than ever. Here we review the significant progress made in the design and creation of a minimal organism. We discuss how comparative genomics, gene essentiality data, naturally small genomes, and metabolic modeling are all being applied to produce a catalogue of the biological functions essential for life. We compare the minimal gene sets from three published sources with functions identified in 13 existing gene essentiality datasets. We examine how genome-scale metabolic models have been applied to design a minimal metabolism for growth in simple and complex media. Additionally, we survey the progress of efforts to construct a minimal organism, either through implementation of combinatorial deletions in Bacillus subtilis and Escherichia coli or through the synthesis and implantation of synthetic genomes.

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Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast

Cited by 275 publications

References 12 publications

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Cloning Whole Bacterial Genomes in Yeast

Building the blueprint of life

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