Chapter 32 : Mining Environmental Plasmids for Synthetic Biology Parts and Devices

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In its most widespread meaning, synthetic biology has been defined as the engineering of biology ( ). There are two sides to this definition. The first deals with fundamental science, as synthesis is the counterpart of analysis, the second leg of any rigorous research endeavor. Synthesizing something by rational assembly of its individual components is, in fact, the ultimate proof of understanding, as the celebrated statement of R. Feynman posthumously declared (“What I cannot create, I do not understand.”). On the other hand, the definition above implicitly announces that biological objects can indeed be engineered for a practical purpose. While in the field of molecular biology the term “genetic engineering” is normally used as a metaphor or an analogy, synthetic biology adopts “engineering” as an authentic conceptual and technical frame for repurposing existing biological entities and for creating new-to-nature properties. The underlying idea is that any biological system can be regarded as a combination of individual functional elements that are comparable to those found in man-made devices. These can be described as wholes of a limited number of components that can possibly be combined in novel configurations to modify existing properties or to create new ones—and so can their biological counterparts. Paramount in this concept is the identification and rigorous description of biological parts (the shortest DNA sequences encoding unique, stand-alone, unambiguous biological functions) and devices (an assembly of parts that runs a specified action with a definite input and output governed by a fixed transfer function). Although parts and devices are the basis of any extant biological system, the angle of synthetic biology is that at least some of them can be excised from their natural context, reformatted to meet some compositional standards, and rewired with a different genetic connectivity to create synthetic systems that bring about novel phenotypic traits in a host organism (often called “the chassis” in the synthetic biology jargon) ( ). For this to happen, it is essential that parts and devices maintain the functions and the parameters that they possess in their natural context once they are placed somewhere else.

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014
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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.

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014
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Image of Figure 2
Figure 2

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.

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014
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Figure 3

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.

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014
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Figure 4

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.

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014
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Table 1

Principal biological parts found in BHR environmental plasmids

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014
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Table 2

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

Citation: Martínez-García E, Benedetti I, Hueso A, de Lorenzo V. 2015. Mining Environmental Plasmids for Synthetic Biology Parts and Devices, p 633-649. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0033-2014

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