Cloning whole bacterial genomes in yeast

Benders, Gwynedd A.; Noskov, Vladimir N.; Denisova, Evgeniya A.; Lartigue, Carole; Gibson, Daniel G.; Assad-García, Nacyra; Chuang, Ronald Y.; Carrera, William; Moodie, Monzia; Algire, Mikkel A.; Phan, Quang N.; Alperovich, Nina; Vashee, Sanjay; Merryman, Chuck; Venter, J. Craig; Smith, Hamilton O.; Glass, John I.; Hutchison, Clyde A.

doi:10.1093/nar/gkq119

Cited by 150 publications

(133 citation statements)

References 63 publications

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“…This estimation was confirmed by the top-down reductive work carried out on the genome of Mycoplasma genitallium, which has the smallest genome known yet (18). This work required the development of previously undescribed cloning methods to reconstitute genome-size DNA plasmids (58)(59)(60). DNA programs of smaller size can be also prepared by recombination or direct assembly of synthesized oligonucleotides (61)(62)(63).…”

Section: Bottom-up Development Of An Artificial Cell: Broad Consideramentioning

confidence: 94%

Development of an artificial cell, from self-organization to computation and self-reproduction

Noireaux¹,

Maeda

Libchaber

2011

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

296

267

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This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.Defining Life Since Schrödinger's classic book What Is Life? (1), the definition of life has always been a puzzle with a variety of partial answers, none really satisfying. One answer is that life is a coded system with mutation and error correction (2). The information is encoded into DNA (the genotype) and the genetic code allows translating this information into proteins (the phenotype). The genetic code is universal, and the genome can support mutations, recombination, duplication, and partial transfer from organisms to organisms. Error correction limits the risk of melting the information into random sequences. This is fine, but life is also metabolism with a permanent absorption and transformation of nutrients from the environment (3). A constant flux of energy is needed to sustain the operation of this living dynamical system out of equilibrium. But life is also self-reproduction (4). Cells originate from preexisting cells by cell division. The DNA genome is replicated, the cell self-reproduces as well as the complete organism. Is self-reproduction a constraint of evolution present at each step and imposing severe design constraints or a possible definition of life? Or is life an emerging phenomenon resulting from complex chemical reactions, developing networks and cycles until, at a certain critical size of this network, life starts as an emerging property (5)? Within this approach, life is a dynamical system where signal to noise is just marginal; a living system positions itself at the edge of chaos. Finally the only generally accepted theme is that life is the result of evolution, natural selection, and adaptation (6), a...

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Section: Bottom-up Development Of An Artificial Cell: Broad Consideramentioning

confidence: 94%

Development of an artificial cell, from self-organization to computation and self-reproduction

Noireaux¹,

Maeda

Libchaber

2011

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

296

267

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“…However, constructing a new cell from the genome of a different bacterium has been done, by inserting the genome in the cell envelope (membrane) of the other. 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).…”

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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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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“…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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Cloning whole bacterial genomes in yeast

Cited by 150 publications

References 63 publications

Development of an artificial cell, from self-organization to computation and self-reproduction

Development of an artificial cell, from self-organization to computation and self-reproduction

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

Building the blueprint of life

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