Gibson, D.G. et al. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc. Natl. Acad. Sci. USA 105, 20404-20409

The J. Craig Venter Institute, Synthetic Biology Group, Rockville, MD 20850, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2009; 105(51):20404-9. DOI: 10.1073/pnas.0811011106
Source: PubMed


We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.

Download full-text


Available from: Gwynedd Benders, Mar 29, 2014
1 Follower
39 Reads
  • Source
    • "As recombinational repair is far more prominent in yeast than ligation or nonhomologous endjoining of a linearized plasmid, the background of nonrecombinant vector is generally very low (Ma et al. 1987; Hudson et al. 1997; Raymond et al. 1999; Tsvetanova et al. 2011). Up to 25 large (17–35 kb) DNA fragments have been assembled efficiently into one vector after cotransformation in yeast without direct selection for any of the fragments except the vector backbone (Gibson et al. 2008b). If a low recombination rate is anticipated, for example owing to the low concentration or large size of an insert, loss of a (counter)selectable marker in the vector can be used to screen for recombinants among the resulting transformants (Ma et al. 1987; Gunyuzlu et al. 2001; Noskov et al. 2002; Raymond et al. 2002; Kitazono 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the past decade, the focus of cloning has shifted from constructing plasmids that express a single gene of interest to creating multigenic constructs that contain entire pathways or even whole genomes. Traditional cloning methods that rely on restriction digestion and ligation are limited by the number and size of fragments that can efficiently be combined. Here, we focus on the use of homologous-recombination-based DNA manipulation in the yeast Saccharomyces cerevisiae for the construction of plasmids from multiple DNA fragments. Owing to its simplicity and high efficiency, cloning by homologous recombination in yeast is very accessible and can be applied to high-throughput construction procedures. Its applications extend beyond yeast-centered purposes and include the cloning of large mammalian DNA sequences and entire bacterial genomes. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 09/2015; 2015(9):pdb.top084111. DOI:10.1101/pdb.top084111 · 4.63 Impact Factor
  • Source
    • "The power of the recombination machinery of Saccharomyces cerevisiae was also recently demonstrated with the successful de novo synthesis of the Mycoplasma genitalium genome. First, large fragments were assembled in vitro, with a method now designated as Gibson Assembly (Gibson et al. 2008a), and the resulting large fragments were assembled in vivo in Saccharomyces cerevisiae using recombination (Gibson et al. 2008b). Last but not least, Saccharomyces cerevisiae has the benefit of having well-developed transformation protocols for efficient introduction of foreign DNA (Gietz and Schiestl 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Carboxylic acids are important bulk chemicals that can be used as building blocks for the production of polymers, as acidulants, preservatives and flavour compound or as precursors for the synthesis of pharmaceuticals. Today, their production mainly takes place through catalytic processing of petroleum-based precursors. An appealing alternative would be to produce these compounds from renewable resources, using tailor-made microorganisms. Saccharomyces cerevisiae has already demonstrated its value for bioethanol production from renewable resources. In this review, we discuss Saccharomyces cerevisiae engineering potential, current strategies for carboxylic acid production as well as the specific challenges linked to the use of lignocellulosic biomass as carbon source.
    Applied Microbiology and Biotechnology 06/2014; 98(17). DOI:10.1007/s00253-014-5866-5 · 3.34 Impact Factor
  • Source
    • "Therefore the in vivo S. cerevisiae recombination method was exploited to complete the final whole genome assembly . In the meantime, it was also found possible to directly assemble 25 DNA cassettes from the earliest stages into a complete genome in a single step by in vivo recombination in S. cerevisiae (Gibson et al., 2008). Two years later, a M. mycoides cell controlled by the chemically synthesized genome was created and exhibited the in silico designed phenotype (Gibson et al., 2010 ). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA assembly is one of the most important foundational technologies for synthetic biology and metabolic engineering. Since the development of the restriction digestion and ligation method in the early 1970s, a significant amount of effort has been devoted to developing better DNA assembly methods with higher efficiency, fidelity, and modularity, as well as simpler and faster protocols. This review will not only summarize the key DNA assembly methods and their recent applications, but also highlight the innovations in assembly schemes and the challenges in automating the DNA assembly methods. This article is protected by copyright. All rights reserved.
    FEMS Yeast Research 06/2014; DOI:10.1111/1567-1364.12171 · 2.82 Impact Factor
Show more