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Mining Environmental Plasmids for Synthetic Biology Parts and Devices

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  • Authors: Esteban Martínez-García1, Ilaria Benedetti2, Angeles Hueso3, Víctor De Lorenzo4
  • Editors: Marcelo Tolmasky5, Juan Carlos Alonso6
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Systems and Synthetic Biology Programs, Centro Nacional de Biotecnología, CNB-CSIC, 3, Darwin Street, 28049 Madrid, Spain; 2: Systems and Synthetic Biology Programs, Centro Nacional de Biotecnología, CNB-CSIC, 3, Darwin Street, 28049 Madrid, Spain; 3: Systems and Synthetic Biology Programs, Centro Nacional de Biotecnología, CNB-CSIC, 3, Darwin Street, 28049 Madrid, Spain; 4: Systems and Synthetic Biology Programs, Centro Nacional de Biotecnología, CNB-CSIC, 3, Darwin Street, 28049 Madrid, Spain; 5: California State University, Fullerton, CA; 6: Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain
  • Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014
  • Received 08 December 2014 Accepted 09 December 2014 Published 20 February 2015
  • Víctor de Lorenzo, vdlorenzo@cnb.csic.es
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  • Abstract:

    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 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.

  • Citation: Martínez-García E, Benedetti I, Hueso A, De Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices. Microbiol Spectrum 3(1):PLAS-0033-2014. doi:10.1128/microbiolspec.PLAS-0033-2014.

Key Concept Ranking

Mobile Genetic Elements
0.736481
DNA Synthesis
0.4844583
Type IV Secretion Systems
0.4137747
0.736481

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/content/journal/microbiolspec/10.1128/microbiolspec.PLAS-0033-2014
2015-02-20
2017-05-27

Abstract:

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 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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FIGURE 1

Conditional replication and suicide delivery device. Replication of some plasmids requires (apart from host factors) the action of a specific replication protein on the vegetative sequence. For the R6K plasmid, it is possible to reshape the natural arrangement in of the gene that encodes the replication protein Π and the so that they are in . In this instance, replication of the resulting plasmid depends entirely on expression of , e.g., from an engineered chromosomal location. On the other hand, plasmids with an can be mobilized into conjugal recipients through the action of the genes of a self-conjugative plasmid (e.g., RK2). By combining the two traits in the same covalently closed circular DNA, one creates a plasmid that is both entirely dependent on a specialized host for replication and can be delivered to a recipient where it can transiently stay but not replicate. This is the basis of the many conditional suicide systems for insertion of transposons that are found in the genetic engineering and synthetic biology literature. doi:10.1128/microbiolspec.PLAS-0033-2014.f1

Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014
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Tripartite matings for plasmid mobilization. The transfer of an -containing plasmid from a donor to a target recipient can be brought about by setting a mating between the two partners (donor and recipient) plus a helper strain that transiently delivers the functions that are necessary for the passage of the plasmid from one to the other. If the replication origin of the thereby transferred plasmid is BHR, its DNA can further proliferate in the non- recipient cells of the triparental mating (case A). The helper plasmid is not inherited in any case, as its replication origin (e.g., ColE1 in this example) is not functional in nonenteric recipients. Alternatively, neither the plasmid of interest nor the helper construct can replicate in the destination strain (case B), thereby providing an excellent scenario for suicide delivery, e.g., for selecting insertions of a transposon borne by the mobilized plasmid. doi:10.1128/microbiolspec.PLAS-0033-2014.f2

Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014
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The basic organization of synthetic biology constructs. Genetic devices are usually composed of a regulatory node that includes a transcriptional factor (an activator or a repressor) responsive to a physicochemical signal (e.g., a chemical inducer) that triggers its ability to stimulate transcription. , gene encoding the regulatory protein; , promoter of the regulatory gene; T, transcriptional terminator; UTR, untranslated regions of mRNA. The output of the regulatory node is a given level of PoPS (polymerase per second), which represents the count of RNAP molecules that pass through the promoter DNA each second. PoPS is then wired to a gene of interest (), which is punctuated by 5′-UTR and 3′-UTR regions. doi:10.1128/microbiolspec.PLAS-0033-2014.f3

Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014
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The Standard European Vector Architecture (SEVA) pipeline. (A) The organization of all SEVA constructs is framed within three basic DNA parts of reference, i.e., two transcriptional terminators and one . (B) The positions of these parts leave three openings flanked by specific and unusual restriction sites which are then used to insert synthetic DNA fragments encoding an antibiotic resistance marker, an origin of replication, and a cargo segment. The orientation and specifications that such segments must follow to acquire the SEVA standard are described at http://seva.cnb.csic.es. (C) Segments are assembled on the formatted frame, and (D) a pSEVA vector is added to the database and to the material repository. doi:10.1128/microbiolspec.PLAS-0033-2014.f4

Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014
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Tables

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TABLE 1

Principal biological parts found in BHR environmental plasmids

Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014
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TABLE 2

Databases, repositories, and suppliers of synthetic biology parts and devices

Source: microbiolspec February 2015 vol. 3 no. 1 doi:10.1128/microbiolspec.PLAS-0033-2014

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