Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Dymond, Jessica S.; Richardson, Sarah M.; Coombes, Candice; Babatz, Timothy D.; Muller, Héloïse; Annaluru, Narayana; Blake, William J.; Schwerzmann, Joy; Dai, Junbiao; Lindstrom, Derek L.; Boeke, Annabel C.; Gottschling, Daniel E.; Chandrasegaran, Srinivasan; Bader, Joel S.; Boeke, Jef D.

doi:10.1038/nature10403

Cited by 411 publications

(433 citation statements)

References 29 publications

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“…In our previous work on synthetic yeast chromosomes (8,9), we have adapted a chemically regulated Cre (27) and shown that it very effectively kills yeast with a semisynthetic genome containing numerous loxP sites flanking a number of essential genes. The specialized Cre protein from ref.…”

Section: Resultsmentioning

confidence: 99%

“…In particular, the booming field of synthetic biology (1) demonstrates the feasibility of de novo synthesis of viral genomes (2)(3)(4)(5), bacterial genomes (6,7), and eukaryotic chromosomes (8,9). The technologies underpinning synthetic biology are advanced DNA synthesis and assembly (10), genome editing (11,12), and computational assisted designs (13)(14)(15), which are all becoming commoditized and thus increasingly available to the public.…”

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confidence: 99%

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Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

Cai

Agmon

Choi

et al. 2015

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

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Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer’s yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational “safeguard” control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10−10), consistent with their orthogonal nature and the individual escape frequencies of <10−6. Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

Cai

Agmon

Choi

et al. 2015

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

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“…The de novo design of cells with synthetic genomes that are viable in a well-defined environment might require only the constitutive expression of the minimal set of genes required for life (26), but design of genomes adapted to various environments requires incorporating computational methodologies evolving GTRNs. We expect that the improvement of GTRNs and the rapid development of technologies allowing the synthesis of novel genomes and their introduction into hosts (27)(28)(29) will allow the construction of simplified genomes.…”

Section: Discussionmentioning

confidence: 99%

Computational design of genomic transcriptional networks with adaptation to varying environments

Carrera

Elena

Jaramillo

2012

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

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Transcriptional profiling has been widely used as a tool for unveiling the coregulations of genes in response to genetic and environmental perturbations. These coregulations have been used, in a few instances, to infer global transcriptional regulatory models. Here, using the large amount of transcriptomic information available for the bacterium Escherichia coli, we seek to understand the design principles determining the regulation of its transcriptome. Combining transcriptomic and signaling data, we develop an evolutionary computational procedure that allows obtaining alternative genomic transcriptional regulatory network (GTRN) that still maintains its adaptability to dynamic environments. We apply our methodology to an E. coli GTRN and show that it could be rewired to simpler transcriptional regulatory structures. These rewired GTRNs still maintain the global physiological response to fluctuating environments. Rewired GTRNs contain 73% fewer regulated operons. Genes with similar functions and coordinated patterns of expression across environments are clustered into longer regulated operons. These synthetic GTRNs are more sensitive and show a more robust response to challenging environments. This result illustrates that the natural configuration of E. coli GTRN does not necessarily result from selection for robustness to environmental perturbations, but that evolutionary contingencies may have been important as well. We also discuss the limitations of our methodology in the context of the demand theory. Our procedure will be useful as a novel way to analyze global transcription regulation networks and in synthetic biology for the de novo design of genomes.automated design | synthetic genomics | genome refactoring | evolutionary computation O rganisms have evolved mechanisms for regulating transcription to better adapt to changing environments. Could such regulation be engineered in a different way (1, 2)? Recent experiments investigating the evolvability of bacterial transcriptional regulatory networks (TRNs) have shown that the massive addition of new links to the network does not significantly alter cell growth. Isalan et al. (3) added transcriptional fusions of promoters with different master transcriptional regulators and showed that Escherichia coli (E. coli) tolerated almost all rewired networks; however, growth was perturbed by as much as 5% (3). This inherent predisposition of E. coli networks to dampen extreme changes in their circuitry enables the possibility of conducting genome-wide rewiring (4). Global transcription regulation could also be analyzed by comparing the regulatory models from distant organisms, provided they show a similar response to the set of studied environments. In this way, they could provide alternative regulatory models, although the lack of knowledge of species-specific selective pressures may blur the conclusions. We will propose here an alternative evolution experiment, which will be conducted computationally thanks to the availability of a quantitative model for the genom...

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“…At the largest scale of DNA assembly, the landmark genome synthesis projects for Mycoplasma genitalium 63 and Mycoplasma mycoides 64 have shown that different scales of assembly require different methods: Gibson assembly can be used to join gene-size DNA fragments in a scale of up to a hundred kilobases, but beyond that in vivo recombination assembly in S. cerevisiae becomes necessary. The global project to construct a synthetic version of the yeast genome also recognises the need for different methods at different scales and utilises combinations of Gibson assembly, USER cloning, traditional digestion and ligation and in vivo recombination to hierarchically combine short DNA fragments into 50 kb synthetic 'megachunks' that replace their equivalent endogenous regions in the genome 65,66 . Given that the work of this project is shared between teams around the world, it is not surprising that standardisation is required to ensure efficient progress.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Cited by 411 publications

References 29 publications

Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes

Computational design of genomic transcriptional networks with adaptation to varying environments

Bricks and blueprints: methods and standards for DNA assembly

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