Combining two genomes in one cell: Stable cloning of the
            <i>Synechocystis</i>
            PCC6803 genome in the
            <i>Bacillus subtilis</i>
            168 genome

Itaya, Mitsuhiro; Tsuge, Kenji; Koizumi, Maki; Fujita, Kyoko

doi:10.1073/pnas.0503868102

Cited by 175 publications

(171 citation statements)

References 39 publications

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“…(40). Reiterative Recombination is one of several techniques that tackle the second issue, integrating exogenous pathways of genes into the chromosome (18)(19)(20). However, Reiterative Recombination is uniquely able to integrate pathways in a highly efficient manner to access large numbers of variant strains.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…The second class of DNA assembly methods exploits in vivo homologous recombination to assemble DNA directly in the cell (16)(17)(18)(19)(20)(21). These techniques are attractive for their simplicity, as they require only a straightforward transformation step.…”

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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“…Holt et al (17) proposed using the lambda Red recombination system to assemble an 1830-kb Haemophilus influenzae genome within an E. coli cell. Itaya et al (18) and Yonemura et al (19) M J01 J02 J03 J04 J05 J06 J07 J09 J11 J12 J13 J14 J15 J17 J18 J19 J20 J21 J22 J23 J24 -c20), and multiplex PCR, with primer set 3, was performed. Clone 11 generated all 10 predicted amplicons and thus was selected for further analysis.…”

Section: Discussionmentioning

confidence: 99%

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

Gibson

Benders

Axelrod

et al. 2008

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

411

318

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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.in vivo DNA assembly ͉ genome synthesis ͉ combinatorial assembly ͉ yeast transformation ͉ Mycoplasma genitalium ͉ synthetic biology Y east has long been considered a genetically tractable organism because of its ability to take up and recombine DNA fragments. More than 30 years ago, Hinnen et al. (1) reported the restoration of leucine biosynthesis in Saccharomyces cerevisiae by transformation of a leu2 -strain to LEU2ϩ using a method involving spheroplasts, CaCl 2 , and PEG. Soon after, Orr-Weaver et al. (2) reported mechanistic studies demonstrating that DNA molecules taken up during yeast transformation can integrate into yeast chromosomes through homologous recombination, and that the ends of the linear-transforming DNA are highly recombinogenic and react directly with homologous chromosomal sequences, whereas circular plasmids carrying yeast sequences integrate by a single crossover and only at low frequency. Subsequently, yeast transformation has become an indispensable tool in yeast genetics.Yeast recombination has since been applied to the construction of plasmids and yeast artificial chromosomes (YACs). In 1987, Ma et al. (3) constructed plasmids from two cotransformed DNA fragments containing homologous regions. In another process, called linker-mediated assembly, any DNA sequence can be joined to a vector DNA using short synthetic linkers that bridge the ends (4, 5). Similarly, four or five overlapping DNA pieces can be assembled and joined to vector DNA (4, 6, 7). This work demonstrated that yeast cells can take up multiple pieces of DNA, and that homologous yeast recombination is sufficiently efficient to correctly assemble the pieces into a single recombinant molecule.The limitations of assembly methods in yeast remain unknown. We recently assembled an entire synthetic M. genitalium genome using a combination of in vitro enzymatic recombination in early stages and in vivo yeast recombination in the final stage to produce the complete genome (8). In the first stage, overlapping Ϸ6-kb DNA cassettes were joined four at a time to form 25 Ϸ24-kb A-series assemblies. Three A-series assemblies were then joined to make 1/8 genome Ϸ72-kb B-series assemblies, and then two Bseries assemblies were assembled to make each of the Ϸ145-kb quarter-genome C-series assemblies. We accomplished the final assembly in yeast using three quarter-genome fragments and a fourth quarter fragment that had been cleaved by a restriction enzyme to provide a site for insertion of the vector DNA. Thus, some individual yeast cells have the capacity to simultaneously tak...

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“…In fact, attempts in addition of DNA into a chromosome are not always successful. When trying to insert the whole Synechocystis PCC6803 chromosome into the B. subtilis chromosome, Itaya et al (2005) were successful only when the combined chromosome was symmetric with respect to the replication origin and terminus. Similarly, chromosomes of E. coli and Haemophilus influenzae were also successfully combined.…”

Section: The Chromosomal Sequence Changes and Negative Consequencesmentioning

confidence: 99%

The Bacterial Chromosomal Sequence and Related Issues

Phan

Nguyễn

2013

American Journal of Virology

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There has been a rapid increasing in the number of bacterial genomes that are completely sequenced recently. However, nucleotide arrangement in bacterial chromosome is still not clear, though it is known that the chromosomal strand is symmetric as a whole but asymmetric locally. The unequal distribution of adenine and thymine as well as cytosine and guanine in the local genomic sequences has been suggested to be associated with transcription and replication. In the bacterial chromosome, distribution of nucleotides in the leading and lagging strands, which interact with the structurally asymmetric DNA polymerase during replication process, has also been reported to be biased. Recent studies on changing the nucleotide sequence of bacterial chromosomes have revealed the importance of nucleotide arrangement in chromosome, which exerts influences on success of the chromosome engineering experiments. This review highlights common properties of bacterial chromosomal sequences with related issues involved in the nucleotide arrangement in chromosome, emphasizing that arrangement of nucleotides in chromosome regarding local strand asymmetry and chromosomal strand symmetry needs to be made clear so as to help in experimental design for effectiveness in changing chromosomal sequences or creating artificial chromosomes.

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Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome

Cited by 175 publications

References 39 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

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

The Bacterial Chromosomal Sequence and Related Issues

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