GeneGuard: A Modular Plasmid System Designed for Biosafety

Wright, Oliver; Delmans, Mihails; Stan, Guy-Bart; Ellis, Tom

doi:10.1021/sb500234s

Cited by 109 publications

(88 citation statements)

References 53 publications

(86 reference statements)

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“…There is also recent work to reduce unintended plasmid propagation in bacteria (22). Existing biocontainment technologies usually depend on a singlecellular mechanism.…”

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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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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“…There is also recent work to reduce unintended plasmid propagation in bacteria (22). Existing biocontainment technologies usually depend on a singlecellular mechanism.…”

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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“…By artificially placing the pir gene in the chromosome and the oriV in a plasmid, one not only ensures a narrowrange replication system, but fulfills the complete requirement of a strain-specific host for vector propagation (18). This feature has been exploited in a large number of synthetic biology circuits aimed at suicide delivery of transposon vectors (19-21) (see below) and engineering of biosafety circuits by designing mutual host-plasmid addiction (22). Not surprisingly, the small collection of narrow host range origins of replication just mentioned have been incorporated into the various repositories of biological parts available to synthetic biologists (23,24) (http://parts.igem.org).…”

Section: Narrow Host Range Origins Of Replicationmentioning

confidence: 99%

“…In this way, any bacterium that loses the plasmid is eliminated. The genes infA (encoding translation initiation factor 1) (54), asd (aspartate-betasemialdehyde dehydrogenase) (55), dapA (dihydrodipicolinate synthase) (22), and thyA (thymidylate synthase) (56) have been used to this end, and many others are surely eligible for the same purpose. This type of marker eliminates the need for drug resistance markers in the vector and is thus appealing whenever stable maintenance of engineered traits is required in the absence of external selection.…”

Section: Selection Markersmentioning

confidence: 99%

“…Although the biological role of colicins is still debated (71), they provide a powerful tool to engineer population-wide circuits for genetic and biological containment of recombinant DNA (72,73). In reality, one of the most sophisticated containment hostplasmid mutual systems designed thus far combines conditional plasmid replication (i.e., initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host), rich-media-compatible auxotrophies (dapA and thyA; see above), and toxin-antitoxin pairs (22). Such constructs ensure a potentially perfect genetic firewall to engineered genetic devices without significantly affecting (at least in the short run) growth parameters.…”

Section: Toxin-antitoxin Systemsmentioning

confidence: 99%

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Mining Environmental Plasmids for Synthetic Biology Parts and Devices

2015

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The scientific and technical ambition of contemporary synthetic biology is the engineering of biological objects with a degree of predictability comparable to those made through electric and industrial manufacturing. To this end, biological parts with given specifications are sequence-edited, standardized, and combined into devices, which are assembled into complete systems. This goal, however, faces the customary context dependency of biological ingredients and their amenability to mutation. Biological orthogonality (i.e., the ability to run a function in a fashion minimally influenced by the host) is thus a desirable trait in any deeply engineered construct. Promiscuous conjugative plasmids found in environmental bacteria have evolved precisely to autonomously deploy their encoded activities in a variety of hosts, and thus they become excellent sources of basic building blocks for genetic and metabolic circuits. In this article we review a number of such reusable functions that originated in environmental plasmids and keep their properties and functional parameters in a variety of hosts. The properties encoded in the corresponding sequences include inter alia origins of replication, DNA transfer machineries, toxin-antitoxin systems, antibiotic selection markers, site-specific recombinases, effector-dependent transcriptional regulators (with their cognate promoters), and metabolic genes and operons. Several of these sequences have been standardized as BioBricks and/or as components of the SEVA (Standard European Vector Architecture) collection. Such formatting facilitates their physical composability, which is aimed at designing and deploying complex genetic constructs with new-to-nature properties.

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“…Although recombinant DNA technology was established more than four decades ago (1), the fact that genetically modified organisms have not caused any substantial deleterious incident is due, in part, to precautionary measures taken by professional genetic engineers and the high cost of genetic engineering, largely restricting its use to academic and industrial laboratories. However, in the current era of "do-ityourself" synthetic biology, with rapid technological advances in DNA synthesis and assembly (2), genome editing (3, 4) and computational tools for design (5-9), and with the decreasing cost of DNA synthesis, the need for genomic safeguards (SGs) is clear.Early biocontainment efforts focused on the use of metabolic auxotrophy dependence (10, 11), toxin/antitoxin-dependent suicide (12-15), or both (16,17). Although top performers using these strategies do comply with the NIH standard for SGs (10…”

mentioning

confidence: 99%

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Agmon

Tang

Yang

et al. 2017

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

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As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial-and yeastbased safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional-and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiaebased transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.genome safety | escape mutants | Rpd3L | histone deacetylase | yeast W ith the increasing use of genetically modified organisms, there is a growing need for biocontainment to secure biosystems from outside malice as well as to prevent propagation in an open system in the event of advertent or inadvertent release to the environment. Although recombinant DNA technology was established more than four decades ago (1), the fact that genetically modified organisms have not caused any substantial deleterious incident is due, in part, to precautionary measures taken by professional genetic engineers and the high cost of genetic engineering, largely restricting its use to academic and industrial laboratories. However, in the current era of "do-ityourself" synthetic biology, with rapid technological advances in DNA synthesis and assembly (2), genome editing (3, 4) and computational tools for design (5-9), and with the decreasing cost of DNA synthesis, the need for genomic safeguards (SGs) is clear.Early biocontainment efforts focused on the use of metabolic auxotrophy dependence (10, 11), toxin/antitoxin-dependent suicide (12-15), or both (16,17). Although top performers using these strategies do comply with the NIH standard for SGs (10

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GeneGuard: A Modular Plasmid System Designed for Biosafety

Cited by 109 publications

References 53 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

Mining Environmental Plasmids for Synthetic Biology Parts and Devices

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

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