Esteban Martınez-Garcıa,1 Ilaria Benedetti,1

Angeles Hueso,1 and Vıctor de Lorenzo1

32Mining Environmental Plasmidsfor Synthetic Biology Partsand Devices

INTRODUCTIONIn its most widespread meaning, synthetic biology hasbeen defined as the engineering of biology (1, 2). Thereare two sides to this definition. The first deals withfundamental science, as synthesis is the counterpart ofanalysis, the second leg of any rigorous research en-deavor. Synthesizing something by rational assemblyof its individual components is, in fact, the ultimateproof of understanding, as the celebrated statement ofR. Feynman posthumously declared (“What I cannotcreate, I do not understand.”). On the other hand, thedefinition above implicitly announces that biologicalobjects can indeed be engineered for a practical pur-pose. While in the field of molecular biology the term“genetic engineering” is normally used as a metaphoror an analogy, synthetic biology adopts “engineering”as an authentic conceptual and technical frame forrepurposing existing biological entities and for creatingnew-to-nature properties. The underlying idea is thatany biological system can be regarded as a combinationof individual functional elements that are comparableto those found in man-made devices. These can be de-scribed as wholes of a limited number of components

that can possibly be combined in novel configurationsto modify existing properties or to create new ones—and so can their biological counterparts. Paramount inthis concept is the identification and rigorous descrip-tion of biological parts (the shortest DNA sequencesencoding unique, stand-alone, unambiguous biologicalfunctions) and devices (an assembly of parts that runsa specified action with a definite input and outputgoverned by a fixed transfer function). Although partsand devices are the basis of any extant biological sys-tem, the angle of synthetic biology is that at least someof them can be excised from their natural context, re-formatted to meet some compositional standards, andrewired with a different genetic connectivity to createsynthetic systems that bring about novel phenotypictraits in a host organism (often called “the chassis” inthe synthetic biology jargon) (3). For this to happen, itis essential that parts and devices maintain the func-tions and the parameters that they possess in their natu-ral context once they are placed somewhere else.

Alas, any biologist knows this is not likely to bethe case (4). Unlike components used in industrial man-ufacturing, the behavior of biological constituents is

1Systems and Synthetic Biology Programs, Centro Nacional de Biotecnologıa, CNB-CSIC, 28049 Madrid, Spain.

Plasmids—Biology and Impact in Biotechnology and DiscoveryEdited by Marcelo E. Tolmasky and Juan C. Alonso

© 2015 American Society for Microbiology, Washington, DCdoi:10.1128/microbiolspec.PLAS-0033-2014

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extremely context-dependent—let alone that they canmutate and evolve erratically. Furthermore, their com-binations may create emergent properties that areoften unpredictable, thereby hindering any rigorous en-gineering based on them. Not surprisingly, many con-temporary synthetic biology efforts try to tackle thischallenge. One of the avenues to this end is the pursuitof “orthogonality” of the engineered constructs in re-spect to their host. This concept, imported from mathe-matics and computer science, indicates zero or aminimal influence of the engineered devices and sys-tems on their biological carrier and vice versa. One ap-proach to orthogonality involves eliminating as manyas possible of the undesired connections that a part ordevice may have in its natural context or to set new in-formation codes that only the designed construct candecipher (5). But one can also ask whether nature hasin some cases favored the evolution of functional mod-ules with basic functions that are to an extent autono-mous in respect to the biological container where theyreside.

This is where plasmids and other mobile genetic ele-ments come into play in the synthetic biology scenario,as they are able to travel through different types of mic-roorganisms and express their genetic complement indiverse genomic and metabolic backgrounds. Phages, inparticular, are programmed to take over the entire ex-pression machinery of the host for its own sake, oftenby means of alternative RNA polymerases (RNAPs)that recognize promoter sequences altogether differentfrom those of the host. Not surprisingly, the RNAP ofbacteriophage T7 (T7 pol) is one of the most used bio-logical parts in synthetic biology: the pair T7pol/PT7(T7 RNAP and the cognate promoter of gene 10 ofphage T7) suffices to trigger transcription of down-stream DNA sequences whether in vivo or in vitro,with the only condition being that nucleoside triphos-phates are available. This happens regardless of the bi-ological carrier, from bacteria to animal cells, thusproviding a reasonably orthogonal tool for manipula-tion gene expression at the user’s will. Also, repressorsfound in transposons are excellent assets for biologicalengineering. Repression is a less demanding mechanismof transcriptional regulation in prokaryotes (6) and isthus far easier to enter in a heterologous context.The tetracycline-dependent TetR repressor of the tetAgene of Tn10 and its target DNA sequence has beenemployed and refactored in many ways to this end (7),and it is also one of the most popular parts for con-struction of artificial genetic circuits. Nevertheless,synthetic biology is not only about heterologous, or-thogonal transcription initiation; it requires many other

engineer-able functions. These are not found in eitherphages or transposons but, as discussed below, onbroad host range (BHR) conjugative or mobilizableplasmids.

PROMISCUOUS PLASMIDS BEARKEY HOST-INDEPENDENTBIOLOGICAL FUNCTIONSBHR conjugative (or at least mobilizable) plasmids areobjectively the most favorable source of natural orthog-onal parts and devices, as long as they deploy their fullor partial genetic complement in diverse biologicalcarriers. The key component to this end is the vegeta-tive origin of replication. It is remarkable that many en-vironmental plasmids seem to have just such an oriVthat either uses the host replication machinery or is ac-companied by a cognate, plasmid-encoded replicationprotein(s) gene(s), with no other recognizable attributeencoded, in what would look like a perfect example of“selfish” DNA. One can consider such minimalist plas-mids as the evolutionary solution to the engineeringchallenge of having an optimal DNA-carrying vector.Note that the bottlenecks that even the simplest plas-mids must overcome for their propagation involve notonly their replication proper (for which they have asuite of mechanisms; see, e.g., references 8, 9) but alsosignaling replication termination and resolution ofreplication intermediates. By default, this can happenthrough homologous recombination between DNA se-quences, but in other instances, specific molecularmechanisms take care of unknotting the otherwise in-tractable replicating molecules.

But just having autonomous replication is no guaran-tee of doing well unless it is accompanied by other com-plementary functions. Environmental plasmids oftenensure their maintenance by having active counterseg-regation systems that force bacteria to retain them orotherwise be killed. But in other cases, stable plasmid-host parterships occur without any conspicuous selec-tive advantage. In many instances, however, plasmidsend up recruiting to their frame some traits that areclearly beneficial to the host. These last include a largesuite of generally dispensable genes which, under cer-tain circumstances, can endow the carrier with somerecompense in return for the physicological load of be-ing carried as an extrachromosomal element. Thesegenes range from resistance to antibiotics (the bestknown plasmid-borne features) to novel metabolic ca-pabilities (e.g., routes for biodegradation of environ-mental pollutants). As discussed below, such pathwaysare themselves a source of both blocks for metabolic

634 PLASMIDS AS GENETIC TOOLS

engineering as well as the source of a wealth of con-ditional expression systems that can be co-opted insynthetic constructs. To assemble whatever circuit ofchoice, such functional DNA fragments are typicallyrecruited to the plasmid frame through a suite of site-specific recombination (SSR) devices which either re-main active in the extant plasmid structures or becomevestigial or even erased after a period of stable coexis-tence with a given host.

In a further turn of the screw, some plasmids deliverto the host novel functions that change their lifestyle(e.g., the thermal window of optimal viability) andeven switch the order of preference of carbon sourceconsumption (10–12). Spreading these properties in apopulation depends on plasmids’ ability to move be-tween different hosts. By default, this can occur throughnatural transformation, but it usually happens throughconjugation. To this end, some plasmids carry a com-plete set of genes for conjugal self-transmission, whileothers just have an origin of transfer (oriT) which canbe recognized by other plasmids’ machinery and thusbring about mobilization. All these components can befound in various configurations in extant plasmidsaccompanied by what we could call “genetic garbage,”

i.e., remnants of insertion sequences and nonfunctionalpseudogenes for which no clear role may be assignedupfront (13).

It is not surprising that some synthetic biologyefforts have been recently directed to re-create a sort ofquintessential BHR plasmid frame, devoid of parasitic-looking elements. Along this line, Hansen (14) con-structed an all-synthetic IncX1 plasmid based on thecomponents just mentioned, although some of themare somewhat compressed in very close or overlappingDNA sequences: tra (conjugal transfer), mob1 (mobili-zation 1), rep/stb (replication initiation/toxin-antitoxinplasmid addiction), mob2 (mobilization 2), par (plas-mid partitioning), and res (resistance marker). The syn-thetic plasmid kept all the properties shared by thenatural members of the IncX1 group but, as long as itwas artificially simplified, created a construct mucheasier to further engineer (15). Table 1 summarizes thekey functions that environmental plasmids are typicallyendowed with and which, following some formattingand repurposing, become useful assets for biosystemengineering along the line of contemporary syntheticbiology. Note that the list is by no means exhaustive,and the reader is directed to the ACLAME database

Table 1 Principal biological parts found in BHR environmental plasmids

Functional segment Description Synthetic biology application References

Origin of replication Narrow Physical assembly, gene cloning in E. coli 16–24Broad Analysis and engineering of diverse Gram-negative

bacteria25–47

Selection marker Antibiotic resistance Plasmid retention, biomarker 49–53Auxotrophy compensation Self-selecting plasmid maintenance 54–56

Toxin/antitoxin systems Plasmid countersegregation Marker-free vectors, biosafety circuits,conditional viability

62–73

Conjugative transfer Origin of transfer Plasmid transmission between E. coli anddiverse recipients

89, 90

Transfer and mobilization Suicide delivery of transposon andtransposon-vectors

91–93

Biological computing and execution of logic(Boolean) programs

94

Regulated promoters Substrate-induced transcription ofmetabolic operons

Regulatory devices turned on by chemical effectors 98–113

Cis-acting antisenseRNAs

Formation of asRNA/target RNAduplexes that are degraded by RNase III

Gene expression silencing 116–123

Metabolic genes Catabolism of unusual carbon sources Models for metabolic engineering andimplant-chassis retroactivity

130, 131

Site-specificrecombination systems

Resolution of replication intermediates Excision of undesired DNA segments in genomesof interest

74–80

Engineering of operative systems, memory,and logic gates

82, 84

Recruitment of functional sequences (Combinatorial) assembly of functional DNA in agene cluster

83, 96

32. MINING ENVIRONMENTAL PLASMIDS FOR SYNTHETIC BIOLOGY 635

(http://aclame.ulb.ac.be) for a thorough compilation ofwhat genes and functions one can find in a large collec-tion of plasmids and other mobile elements. In the fol-lowing sections we discuss in more detail some of theseparts and how they have been incorporated into thesynthetic biology toolbox.

NARROW HOST RANGE ORIGINSOF REPLICATIONPlasmid vectors are the basis of recombinant DNAtechnology and the workhorses of any genetic engineer-ing or synthetic biology project. The one key functionthat enables their existence as extrachromosomal ele-ments is their origin of replication. Most of thoseemployed for gene cloning are intended to work inEscherichia coli and thus have a narrow host range.The most used to this end are those based on the vege-tative origin of replication (oriV) of plasmid ColE1/pMB1, which is the type of default origin of replicationfound in a large number of plasmid vectors (16). Themain advantage is its small size and its mechanism ofreplication, which is entirely enabled by host functions(i.e., E. coli). This makes the DNA sequences neededfor autonomous propagation of plasmids with a ColE1origin compressed in a very small fragment that canbe easily moved according to the user’s needs. MostColE1-derived vectors bear variants of this oriV withmutations that cause a very high copy number. Whilethis eases DNA extraction and preparation from hostcells, it may become detrimental if the plasmid carriescloned genes—and more so if they are toxic. A secondtype of narrow host range vectors are those based onthe p15A replicon (16). These have a lower copy num-ber than their ColE1 counterparts (but still high) andare compatible with them, thereby enabling expressionof cloned genes from two coexisting vectors. AnotheroriV quite popular in plasmids for E. coli is SC101,whose advantage is its low copy number. The downsidein this case is its lower yield when extracted from hostbacteria and the need for an extra plasmid-encodedreplication protein, which increases the size of theDNA required for its autonomous propagation.

Finally, one narrow host range replication originthat has been heavily refactored for different purposessince its description (17) is that of plasmid R6K. Theproperty that makes this system so attractive is thatthe plasmid-borne replication protein Π (encoded bythe so-called pir gene) can also actuate in trans on theorigin of replication (oriVR6K). This allows the physi-cal separation as well as the mutual dependence ofthe two components (pir gene and oriV) for plasmid

functioning (Fig. 1). By artificially placing the pir genein the chromosome and the oriV in a plasmid, one notonly ensures a narrow-range replication system, butfulfills the complete requirement of a strain-specifichost for vector propagation (18). This feature has beenexploited in a large number of synthetic biology circuitsaimed at suicide delivery of transposon vectors (19–21)(see below) and engineering of biosafety circuits bydesigning mutual host-plasmid addiction (22). Not sur-prisingly, the small collection of narrow host rangeorigins of replication just mentioned have been incor-porated into the various repositories of biological partsavailable to synthetic biologists (23, 24) (http://parts.igem.org).

BHR ORIGINS OF REPLICATIONAs long as synthetic biology projects go beyond modelE. coli strains and toy circuits and enter deploymentof deeply engineered traits for industrial, medical, andenvironmental settings, new hosts (i.e., chassis) differ-ent from E. coli are required, and BHR plasmid vectors

Figure 1 Conditional replication and suicide delivery device.Replication of some plasmids requires (apart from hostfactors) the action of a specific replication protein on the veg-etative oriV sequence. For the R6K plasmid, it is possible toreshape the natural arrangement in cis of the pir gene thatencodes the replication protein Π and the oriV so that theyare in trans. In this instance, replication of the resultingplasmid depends entirely on expression of pir, e.g., from anengineered chromosomal location. On the other hand, plas-mids with an oriT can be mobilized into conjugal recipientsthrough the action of the tra/mob genes of a self-conjugativeplasmid (e.g., RK2). By combining the two traits in the samecovalently closed circular DNA, one creates a plasmid that isboth entirely dependent on a specialized host for replicationand can be delivered to a recipient where it can transientlystay but not replicate. This is the basis of the many conditionalsuicide systems for insertion of transposons that are found inthe genetic engineering and synthetic biology literature.doi:10.1128/microbiolspec.PLAS-0033-2014.f1

636 PLASMIDS AS GENETIC TOOLS

become mandatory. BHR origins of replication havebeen known since the onset of the recombinant DNAera (25, 26), but their generally more complex replica-tion mechanism (and therefore their larger size) havelimited their use in vector development. Such originsafford autonomous propagation of the replicon on avariety of bacterial carriers. Most BHRs of this kindhave been developed for Gram-negative microorga-nisms, although some of them could also be useful inGram-positive bacteria (see below). One widely usedBHR origin is that of plasmid RSF1010. Although itsreplication mechanism is somewhat intricate and thecorresponding functional segment contains intermingledreplication and transfer functions (27, 28), RSF1010-based plasmids are among the most host-promiscuousvectors. The segment of the natural plasmid thatsuffices for autonomous replication has been recentlystreamlined (29) and formatted as a separate modulefor vector assembly (23). A second origin very fre-quently found in BHR vectors is that of the RK2/RP4plasmid (30). Despite the (comparatively) small size ofthe cognate functional segment, the RK2 origin alsocounts among the most promiscuous. A large number ofvectors—before and after the onset of synthetic biology—based on RK2 have been instrumental in analyzingand constructing complex phenotypes in bacteria suchas Pseudomonas, Rhizobium, Agrobacterium, and asuite of Gram-negative species (31–33).

But other origins of replication are increasinglyused for BHR vector development as well. For instance,pBBR1, a plasmid first isolated from Bordetella bron-chiseptica (34), was adopted as the basis of a consider-able number of genetic tools (24, 35–37). One of theadvantages is that BBR1-derived plasmids generallyhave a higher copy number than their RK2-based andRSF1010-based counterparts (with all the pluses andminuses discussed above). By the same token, the originof environmental IncW plasmid pSa (38), which wasadopted in the late 1980s for constructing BHR vectorsfor Agrobacterium (39) and Xanthomonas (40), hasbeen recently reshaped and formatted as a buildingblock for generating standardized vectors for Ralstonia(41). In contrast, genus-specific (e.g., Pseudomonas) ori-gins of replication from, e.g., Pseudomonas savastanoi(42) which were proposed at some point for generatinga suite of new BHR vectors (43) never made it to main-stream use. One way of broadening the range of suchotherwise narrow-range plasmids is to join origins thatfunction alternatively in two or more hosts in the sameDNA segment, as has been done with the originallyP. aeruginosa-only plasmid pRO1614 (44, 45) and theE. coli/Pseudomonas putida/Bacillus shuttle named

pEBP (46). However, despite some incursions into otherfunctional origins of replication, vectors based onRSF1010, RK2, BBR1, and pSa have been the pillars ofthe genetic engineering of Gram-negative organismsother than E. coli for a long time, and now they remainthe basis of standardized modules for assembly of syn-thetic devices for the same type of organisms (23, 24)(http://parts.igem.org). Alas, the same standardizationeffort has been more limited thus far in the Gram-positive realm, although some exciting developmentsare under way at the time of writing this article(47, 48).

SELECTION MARKERSAntibiotic resistance genes have been systematically thefavorite resource for enabling selection of recombinantplasmids. By adding the cognate agent to the selectionmedium, one can not only pick out transformants afterDNA ligation or assembly but also ensure retention ofa given plasmid if no endogenous countersegregationsystem is in operation. A variety of resistance genes forampicillin (bla, Ap) tetracycline (tet, Tc), chloramphen-icol (cat, Cm), kanamycin (kan, Km), streptomycin/spectinomycin (Sm/Sp), and gentamycin (Gm) havebeen retrieved repeatedly from their original naturalcontext (often a clinical isolate) and reused in a largevariety of vectors (49, 50). More recently, their corre-sponding sequences have been formatted as modulesfor assembling all types of synthetic biology constructs(23, 24) (http://parts.igem.org). But the ease of use ofthese markers has two major drawbacks for applica-tions involving industrial or environmental settings.First, the products of these genes act on antibiotics thatare in clinical use (or are similar to them), and engi-neering bacterial biomass which carries resistance genesmay thus raise safety concerns (51). Second, antibioticsthemselves are expensive, and using them as a bulk ad-ditive to any large-scale growth medium might be eco-nomically prohibitive.

This has prompted the quest for alternative markersor strategies to ensure stable plasmid maintenancewithout having to use typical antibiotics. One suchstratagem is the adoption of resistance to herbicides(e.g., bialaphos) (52) or heavy metals (e.g., tellurite)(53) as markers. Another useful approach is placing inthe plasmid the sequence of an essential gene thatcomplements a deletion of the same gene excised fromthe chromosome of the host strain. In this way, anybacterium that loses the plasmid is eliminated. Thegenes infA (encoding translation initiation factor1)(54),asd (aspartate-beta-semialdehydedehydrogenase)

32. MINING ENVIRONMENTAL PLASMIDS FOR SYNTHETIC BIOLOGY 637

(55), dapA (dihydrodipicolinate synthase) (22), andthyA (thymidylate synthase) (56) have been used to thisend, and many others are surely eligible for the samepurpose. This type of marker eliminates the need fordrug resistance markers in the vector and is thus ap-pealing whenever stable maintenance of engineeredtraits is required in the absence of external selection.An alternative approach to ensure recombinant plasmidretention without antibiotics relies on the refactoring oftoxin-antitoxin systems (see below). In addition, userscan consider the definite insertion in the chromosomeof the DNA segment of choice by using one of themany mini-transposon vectors based on either Tn5 (20,21) or Tn7 (57, 58) that have been designed to that endin recent years. Some of these transposon vectors incor-porate in their design the possibility of removing theantibiotic markers employed for selection of insertionevents once transposition has occurred (59, 60).

TOXIN-ANTITOXIN SYSTEMSAlthough many naturally occurring plasmids, in partic-ular the smallest, seem to be maintained on the sheerbasis of their high numbers, others with lower copydoses have developed sophisticated molecular mecha-nisms to coordinate chromosomal and plasmid replica-tion and/or to penalize cells that may accidentally losethe extra replicon. These so-called plasmid addictionsystems (61) seem to be a special case of a more generalmethod for protecting the integrity of the genomiccomplement under environmental and nutritional stressconditions (62–64). These devices systematically in-volve a DNA segment that encodes a toxin and its anti-dote. Toxin-antitoxin modules usually are composed oftwo cognate proteins with different expression levelsand/or half lifetimes (e.g., the parD locus encodingKis/Kid proteins in plasmid R1) (65). Under normalconditions, the antidote inhibits the toxin, whereasplasmid loss makes proteolytic degradation of the anti-dote faster, titrating the remedy out and killing the hostcells. Alternatively, the antidote of the toxin-antitoxinpair is an RNA that prevents translation of a toxic genein such a way that cells that lose the plasmid are ac-tively eliminated by the system. The archetypal exam-ple of such a scenario is that of the parB system of R1,which encodes two small genes, hok and sok (66). Theexpression of the first (a toxin) is regulated by the com-plementary hok antisense RNA to the hok mRNA.Postsegregation killing in single cells is brought aboutby the differential decay of the hok and sok RNA. Themodularity of the system stabilizes any plasmid that isinserted with a parB-encoding DNA segment (67). This

feature has made the parB system a remarkable tool forensuring plasmid maintenance in individual bacteriawithout any external selection (68).

A different type of toxin-antitoxin system is that ofcolicins (69, 70), large protein poisons that are releasedinto the medium to kill related bacteria which mayhave lost the colicin locus (i.e., the DNA segmentencoding the structural gene for the toxin and a colicinimmunity gene) or similar strains that have a receptorfor the colicin in question. Although the biological roleof colicins is still debated (71), they provide a powerfultool to engineer population-wide circuits for geneticand biological containment of recombinant DNA (72,73). In reality, one of the most sophisticated contain-ment host-plasmid mutual systems designed thus farcombines conditional plasmid replication (i.e., initia-tors for the R6K or ColE2-P9 origins are providedin trans by a specified host), rich-media-compatibleauxotrophies (dapA and thyA; see above), and toxin-antitoxin pairs (22). Such constructs ensure a poten-tially perfect genetic firewall to engineered geneticdevices without significantly affecting (at least in theshort run) growth parameters.

SITE-SPECIFIC RECOMBINATIONWhile homologous recombination typically requires aconsiderable degree of DNA identity through a goodlength of the sequences in question, SSR occurs be-tween shorter segments sharing limited similarity. Thisphenomenon, which is widespread through all biolo-gical kingdoms, is brought about by the action of theso-called site-specific recombinases on short DNA siteswhich they manage to recognize, cleave, exchange be-tween DNA strands, and rejoin. This basic device,which by default just needs a recombinase and a suiteof target sites, is a key component of a large collectionof biological phenomena in both eukaryotic and pro-karyotic organisms. One of these functions is the reso-lution of replication intermediates in plasmids with anactive partitioning mechanism. An SSR which has beenrepeatedly reshaped and repurposed for genetic engi-neering is that of the multimer resolution system (mrs)of BHR plasmid RP4. This system is one of the func-tions encoded within the par region, which determinesthe major RP4 stabilization system. The product ofthe parA gene is a site-specific resolvase that cata-lyzes recA-independent intramolecular recombination be-tween two directly oriented resolution (res) sites. If thesesites flank a supercoiled DNA segment, the result of theprocess is the excision of the intervening sequence.Transient expression of parA has been exploited to

638 PLASMIDS AS GENETIC TOOLS

delete specific DNA segments (e.g., antibiotic resistancegenes) delivered with transposons to the chromosomeof target bacteria (e.g., E. coli or Pseudomonas) (74).This permits the retention of heterologous DNA seg-ments devoid of any phenotypic marker to the genomeof the target bacterium, an important considerationfor microorganisms destined for environmental release(75). Further plasmid-based site-specific recombinationsystems have been extensively employed for the sameor similar purposes, e.g., the Cre-lox resolvase of thephage/plasmid P1 (76, 77). But others are availableand well suited as tools for deep refactoring of bothprokaryotic and eukaryotic systems. Among them is theβ-recombination system of pSM19035 (78), a virtuallyorthogonal SSR that originates in the complex repli-cation mechanism of this plasmid of Gram-positivebacteria (79, 80).

Integrons are a second type of useful SSR systemoften found in environmental plasmids and other mo-bile elements (81). They are DNA elements with anability to capture genes with a mechanism that typi-cally involves an integrase gene (int) and a nearby re-combination site (att). The recruitable DNA is locatedon gene cassettes in which the functional sequences areadjacent to a matching att recombination site. The es-sence of the integron is that such DNA sequences mustoccur in covalently closed circular DNA but cannot bereplicated as such; they can only be replicated when re-combination occurs between the two att sites, therebyentering the cassette into the cognate target. As the attsites are maintained after the integration, the resultingmolecular product can again act as a landing pad forfurther integration rounds. Furthermore, this is a re-versible process, so that DNA sequences can be furtherexcised and integrated in a very dynamic fashion. Al-though the activity of integrons is one of the mostdreaded mechanisms of acquisition of antibiotic resis-tance in clinical settings (81), they provide an extra-ordinary platform for creating genetic diversity insynthetic biology constructs (82). In one case, a trypto-phan production operon was reconstructed by sepa-rately delivering individual genes as recombinationcassettes to a synthetic integron platform in E. coli.Integrase-mediated recombination generated a largenumber of genetic combinations in vivo with a largenumber of combinations of thereby arrayed genes,some with increasing production of the amino acid by11-fold compared to the natural gene order of the samepathway (83).

In a further twist of their natural properties, SSRsystems can not only be used as secondary tools forconstruction of synthetic biology devices but also can

be engineered as actuators able to record informationand implement a gene expression program. For in-stance, by flanking transcriptional terminators or pro-moters with SSR targets one can engineer complexcircuits based on the control of the flow of RNAPalong DNA. Integrase-mediated inversion or deletionof such transcription checkpoints allows constructionof AND, NAND, OR, XOR, NOR, and XNOR logicgates that use common signals and enable design of se-quential logic operations (84). Similarly, it is possibleto assemble genetic circuits that use recombinases forrunning Boolean logic functions by means of DNA-encoded memory of events (85). One appealing possi-bility in this context is the engineering of circuits inwhich stable gene expression (output) is the result oftransient environmental or chemical inputs (86). If onecombines SSR-based gates with suitable reporter sys-tems (e.g., the green fluorescent protein gene [GFP])and PCR, it becomes possible to interrogate the state ofthe synthetic devices for past events.

CONJUGATION ANDTRANSFER FUNCTIONSPlasmids are the main agents of horizontal gene trans-fer in nature. This can occur through natural trans-formation (sometimes electro-transformation due toatmospheric lighting) (87, 88), but usually plasmidtransit from one bacterium to another occurs throughconjugation. This phenomenon includes different mech-anisms of DNA delivery that require a physical contactbetween a donor and a recipient, often facilitated byspecific pili which bridge and bring together matingpartners. Complete conjugation systems share a basiclayout composed of four constituents (89). The first isthe so-called relaxase, the key protein in conjugation,which recognizes the second component: a short DNAsequence that acts as the oriT. Having a relaxase andan oriT in cis or in trans generally suffices for a plas-mid to be conjugally transmissible (Fig. 1). The relax-ase catalyzes (i) a site-specific cleavage of the oriTsequence in the donor, thereby producing a DNAstrand that will be transferred, and (ii) the eventualreligation of DNA once it reaches the recipient bacteriaand transfer is completed, thereby reconstituting thewhole plasmid in the new host. Depending on the plas-mid in question, the relaxase may work in concert withother proteins that form a complex called relaxosome.To complete the conjugation process, one needs twoadditional functions. One is the formation of a matingchannel through which DNA can pass from one cell tothe other. This structure consists of a type IV secretion

32. MINING ENVIRONMENTAL PLASMIDS FOR SYNTHETIC BIOLOGY 639

system (T4SS), which transports the relaxase attachedto the DNA to be transferred. The second is the so-called type IV coupling protein (T4CP), which makesthe essential connection of the relaxosome/DNA com-plex and the transport channel. In addition, some con-jugative plasmids encode structural and regulatorygenes for formation of pili on the cell surface, whicheases and tightens the physical connection between thedonor and the recipient.

Typical self-transmissible environmental plasmids(e.g., RK2/RP4 or R388) encode the entire complement(oriT/tra/mob genes) for all functions necessary tobring about the complete transfer process. Other plas-mids may carry only the relaxosomal components (i.e.,oriT, the relaxase gene, and auxiliary proteins). Inthis case (e.g., RSF1010) the DNA can initiate its owntransfer but has to be complemented by functionsof other conjugative plasmids for finishing the task.Finally, other plasmids may just bear DNA sequencesthat can be recognized by relaxases encoded by otherreplicons and used as oriT for lateral transfer.

Some components of the conjugation and mobiliza-tion machineries just mentioned have been repurposedfor diverse genetic engineering and synthetic biologyapplications. The most salient involves the inclusion ofthe RK2/RP4 oriT in a variety of plasmid vectors formaking them mobilizable to new hosts by means ofthe transfer functions of the same plasmid providedin trans (Fig. 1). Note that this conjugative system isthe most promiscuous known, as it is able not only tomove plasmids among bacteria, but also to deliverthem to yeasts and mammalian cells (90). Such a broadcapability has been formatted in various ways. The firstmethods to mobilize oriT-containing plasmids from adonor host (typically E. coli) to other Gram-negativerecipient bacteria required the action of a third matingpartner which transiently provided such tra functionsto the donor but could not replicate in the final recipi-ent (91). The products encoded by the helper plasmidcould thereby escort the transfer of the oriT constructwithout itself being inherited in the destination strain(Fig. 2). In a subsequent advance, specialized strains

Figure 2 Tripartite matings for plasmid mobilization. The transfer of an oriT-containingplasmid from a donor to a target recipient can be brought about by setting a mating betweenthe two partners (donor and recipient) plus a helper strain that transiently delivers the func-tions that are necessary for the passage of the plasmid from one to the other. If the oriV rep-lication origin of the thereby transferred plasmid is BHR, its DNA can further proliferate inthe non-E. coli recipient cells of the triparental mating (case A). The helper plasmid is notinherited in any case, as its replication origin (e.g., ColE1 in this example) is not functionalin nonenteric recipients. Alternatively, neither the plasmid of interest nor the helper constructcan replicate in the destination strain (case B), thereby providing an excellent scenario forsuicide delivery, e.g., for selecting insertions of a transposon borne by the mobilized plasmid.doi:10.1128/microbiolspec.PLAS-0033-2014.f2

640 PLASMIDS AS GENETIC TOOLS

were designed such that the tra functions of RP4 wereintegrated in the chromosome of a donor E. coli strainwhere the oriT-plasmids could be placed and poised forconjugation with available target bacteria (92). Thisallowed users to get the desired plasmid transfer by set-ting a simple two-partner mating (rather than the tri-parental mating above). A side benefit of such a schemewas the possibility to combine plasmid mobilizationwith conditional replication of the same plasmid (possi-ble in the donor bacterium, not possible in the recipi-ent) as a way to deliver transposons engineered in thesame circular DNA frame (18). Alas, the genetic tech-nology available at the time obliged the donor bacteriato carry the Mu phage also (as a carrier of the RP4 trafunctions), thus creating a chance of artifactual inser-tions and misleading phenotypes. Only recently hasthis problem been completely solved by engineering anE. coli Mu-free donor strain that stably harbors all ofthe RP4 conjugative functions and sustains replicationof Π-dependent suicide vectors (93) (see above).

However, conjugation has been exploited in syn-thetic biology not only for the sake of using its func-tional parts, but also as a phenomenon that can beutilized for biological computing and digital cell-to-cellcommunication (94). One can see DNA transfer as ascenario where individual bacterial types perform spe-cific subtasks, the results of which are then communi-cated to other cell types for further processing. Specificcell-cell conjugation allows direct transfer of genetic in-formation in a digital fashion; i.e., cells in a populationcan only have or not have a given plasmid. Mixing dif-ferent strains in a single population allows a multicellu-lar pool to execute the Boolean XOR function. Modelspredict this approach to be more powerful than otherbiological computation approaches using, e.g., quorumsensing. A conjugation-wired computing system couldthus become a realistic methodology to implementnew-to-nature properties in biological systems (94).

To finish this section we need to refer to the so-called integrative and conjugative elements (ICEs),which combine interesting properties of transfer sys-tems and site-specific recombination in the same DNAsegment. ICEs are mobile genetic elements that by de-fault stay in the host chromosome but which can occa-sionally be activated to adopt a plasmid-like form thatcan be transferred between cells by conjugation (95).ICEs can encode genes for antibiotic resistance, meta-bolic functions, virulence factors, and many otheradvantageous traits. The organization of ICEs variesenormously, but inter alia they encode an integrase thatplays a role in different steps of the element’s life cycle.One interesting ICE specimen is the so-called ICEclc,

an element found in Pseudomonas knackmussii B13that provides the host with the capacity to metabolizechlorocatechols (95). The integrase activity of this ICEhas been recently used for developing a genetic toolable to stably implant large DNA fragments into oneor more specific sites of a target chromosome (96).The genetic kit to this end involves only a cloning vec-tor and a mini-transposable element with which largeDNA segments engineered in E. coli can be tagged withthe integrase gene. The system has been instrumental inintroducing cosmids and bacterial artificial chromo-some DNAs from E. coli into P. putida in a site-specificmanner. These developments illustrate how fundamen-tal knowledge of the different steps and components ofhorizontal gene transfer (HGT) become a treasure troveof building blocks for biosystems engineering.

EXPRESSION SYSTEMSEnvironmental plasmids found in bacteria that inhabitniches with a history of pollution by industrial chemi-cals frequently carry in their DNA one or more geneclusters that expand the range of compounds that canbe used as carbon sources (97). It is often the case thattranscription of such genes responds to either the sub-strates of the corresponding pathways or to some oftheir metabolic intermediates. The bottom line is thatin the very competitive environments where these bac-teria thrive, expression of extra genes must be tightlycontrolled for being triggered only when and wherethey are needed. This scenario is thus an excellentground in which to mine pairs of transcriptional fac-tors (TFs)/cognate chemical inducers which can be re-formatted for creating new conditional expressionsystems (98). This approach is helping to overcomethe dearth of alternative inducible devices for syntheticbiology circuits (Fig. 3), which is thus far limited tothose typically used in E. coli: LacI/Plac (and its manyvariants), the thermo-inducible cI-repressed λ phagepromoters, AraC/PBAD, TetR/Ptet (24), and more re-cently, the RhaR/Prha system (99), either by themselvesor in combination with the T7 RNAP.

A number of alternative effector-triggered systemsfrom various origins have been assembled for utiliza-tion in a variety of hosts including (but not limited to)E. coli. The basic expression node includes a low-activitypromoter that transcribes the effector-responsive TF geneand the target cognate promoter that is activated by theTF/inducer complex. Promoters regulated by TFs of thiskind endowed with various degrees of standardizationinclude AlkS/PalkB (activated by n-octane) (100, 101),NahR/Psal (activated by salicylate) (102), CprK/Pcpr

32. MINING ENVIRONMENTAL PLASMIDS FOR SYNTHETIC BIOLOGY 641

(activated by o-chlorophenylacetic acid) (103), ChnR/PchnB (activated by cyclohexanone) (104), and MekR/PmekA (activated by methylethyl ketone) (105). Thebenzoate-inducible pair XylS/Pm, which stems fromthe toluene/m-xylene degradation plasmid pWW0 ofP. putida, has been the basis of a large number ofrecombinant expression systems both in plasmids (106–108) and in transposon vectors (60, 102). It is note-worthy that the first rationally engineered feed-forwardloop described in the literature was built on the basisof genetically rewiring two salicylate-responsive regu-lators for forming a transcriptional amplification cas-cade (109, 110). Another regulator of the pWW0plasmid, the m-xylene-responsive TF called XylR, hasalso been adopted alone or in combination with the T7RNAP for engineering whole-cell biosensors of aro-matic compounds (111–113).

The list of chemically inducible TFs that can beexploited to the same or similar ends is very large(114, 115) and likely to be expanded in the near future.An added bonus of these expression systems is thatowing to being encoded by promiscuous plasmids,they are likely to be operative in a large variety ofhosts. Furthermore, when the devices are placed in ahost without the corresponding metabolic genes, thenthe biochemical network of the recipient is usuallyblind to the effector, and the induction method hasminimal or no impact (i.e., is orthogonal) on the hostphysiology.

Apart from transcriptional regulators, plasmids oftenalso encode antisense RNAs (asRNAs) involved in vari-ous functions that can be recruited for engineering post-

transcriptional circuits and expression systems based onRNA (116–118). To this end, a large number of cis-encoded asRNAs can be found in plasmids, phages,and transposons (119). Some of these asRNAs havebeen repurposed for engineering gene silencing (120,121) and artificially halting bacteriophage infections(122, 123).

REPOSITORIES OF PLASMID-RELATEDPARTS AND DEVICES: THESEVA COLLECTIONOne of the key synthetic biology tenets is the adoptionof rules for the physical (and, wherever possible, func-tional) composition of biological parts to create deviceswith clear boundaries and defined input-output trans-fer functions (124). The advantages, downsides, andchallenges of this view have been extensively discussedelsewhere (4) and will not be addressed here. Differentsynthetic biology communities have adopted differentstrategies for building increasingly complex geneticconstructs on the basis of their own repositories of bio-logical components. Table 2 shows a nonexhaustive listof web resources where some of the most popularmaterials available for synthetic biology purposes arecompiled. Note that each collection has a different de-gree of curation and reliability and also a very diverseretrieve policy—from complete open access to pay-per-request to intricate material transfer agreement (MTA)procedures. One of the most accessed is the Registry ofStandard Biological Parts (http://parts.igem.org). Thiscollection, founded in 2003 at MIT, has accumulated a

Figure 3 The basic organization of synthetic biology constructs. Genetic devices are usuallycomposed of a regulatory node that includes a transcriptional factor (an activator or arepressor) responsive to a physicochemical signal (e.g., a chemical inducer) that triggersits ability to stimulate transcription. R, gene encoding the regulatory protein; PR, promoterof the regulatory R gene; T, transcriptional terminator; UTR, untranslated regions ofmRNA. 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 eachsecond. PoPS is then wired to a gene of interest (GOI), which is punctuated by 5´-UTR and3´-UTR regions. doi:10.1128/microbiolspec.PLAS-0033-2014.f3

642 PLASMIDS AS GENETIC TOOLS

very large number of parts and devices that have beenemployed in educational synthetic biology projects(iGEM: http://igem.org). Although the collection ismostly used and produced by undergraduate students,and quality control is not optimal, the large number ofitems and their inclusion as standardized parts hasmade the registry a very valuable resource for manysynthetic biology endeavors (125–128).

The Standard European Vector Architecture data-base (SEVA-DB) deserves a separate comment (Fig. 4).This platform is both a web resource and a compendi-um of standardized and modular plasmid vectors. Thiscollection originated in our efforts to expand the realmof synthetic biology toward Gram-negative bacteriadifferent from E. coli but endowed with a biotech-nological or environmental value. The idea and designbehind this plasmid vector compilation is simple buthas strict rules. This repository is composed of vectorsthat have up to four different modules decoratinga fixed plasmid backbone. The rules of the SEVA

compositional standard are as follows: (i) the DNAelements that comprise the plasmids have to be devoidof certain restriction sites (for specific details see refer-ence 23; http://seva.cnb.csic.es), and (ii) the differentmodules have to be flanked by other sets of rare restric-tion sequences. In brief, the plasmid backbone is com-mon to all constructs and includes an origin of transfer(oriT) and two transcriptional terminators (T1 and T0)to keep the adjacent DNA segments transcriptionallyinsulated. A typical plasmid from the collection iscomposed of an antibiotic resistance marker module,an origin of replication segment, and a cargo part. Op-timally, vectors may also carry a genetic gadget to en-dow plasmids with additional properties. The antibioticmodule comprises the six most common antibiotic re-sistance markers, and the origin of replication can beone out of nine choices with different functionalitiesin terms of host range (broad and narrow) and copynumber (low, medium, and high copy), leaving theiroptimal combination to the user’s choice. The cargo

Table 2 Databases, repositories, and suppliers of synthetic biology parts and devices

Database/repository webpage Description/application

Public/open source/nonprofithttp://parts.igem.org The largest collection of standardized synthetic biology parts and devices.

Not a curated repositoryhttp://biobricks.org Large number of resources for the open source synthetic biology communityhttp://seva.cnb.csic.es Curated repository of standardized plasmid vectors for engineering non-E. coli bacteriahttp://www.addgene.org The largest distributor of vectors, plasmids, and cloned genes to the academic communityhttp://www.shigen.nig.ac.jp Cloning vectors for E. coli, preserved as purified DNA sampleshttp://bccm.belspo.be Strains, plasmids, libraries, and other biological materials contributed by

Belgian researchershttp://www.bioss.uni-freiburg.de/toolbox Curated collection of vectors (mostly plasmids) for biosystems engineeringhttp://www.science.co.il Compilation of databases of mobile genetic elements, plasmids, and vectorshttp://dnasu.org Repository for plasmid clones and collections with links to collections of

individual researchershttps://public-registry.jbei.org Software and platform for managing information about biological parts and host strainshttp://www.beiresources.org Biological materials and tools with emphasis on infectious diseases

Commercial/privatehttps://www.snapgene.com Software for simulation and guiding of DNA cloning in different types of vectorshttp://cms.plasmidfactory.com/en/ Plasmid vectors made a la carte for specific bioengineering projectshttp://www.lgcstandards-atcc.org ATCC-linked repository of biological materials including vectors and strainshttp://www.lifesciences.sourcebioscience.com Tailored DNA clones, vectors, and bioinformatic serviceshttp://www.promega.es/products/vectors Large collection of vectors and other genetic tools for gene expression and

reporter systemshttp://www.bioclone.us Commercial provider of molecular tools for optimization of gene expressionhttp://plasmid.med.harvard.edu/PLASMID/ Harvard-based provider of sequence-verified plasmid constructshttps://www.neb.com Materials and general support for iGEM projects following standardized

assembly methodshttp://www.lifetechnologies.com Chemical synthesis, cloning, and sequence verification of genetic sequences a la cartehttp://www.genscript.com Gene synthesis, DNA libraries, and building blocks for synthetic biologyhttp://www.labguru.com/features/molecular/plasmids

Plasmid vector database along with tools for cloning projects and plasmid retrieval

32. MINING ENVIRONMENTAL PLASMIDS FOR SYNTHETIC BIOLOGY 643

endows functionalities to the plasmid, e.g., by meansof a polylinker, different promoter-probe elements, ordifferent expression systems. Since modules are punctu-ated by uncommon restriction sites, their exchange forcreation of different plasmid combinations is straight-forward. The collection is updated in real time and isincreasingly adopting the synthetic biology open lan-guage (SBOL) for describing all vectors of the SEVA re-pository in a language that can be interfaced with otherplatforms and understood by robotic DNA synthesisplatforms (129). While the manifest destiny of geneticconstructs is having them made through chemical syn-thesis (for which assembly vectors and material DNArepositories may not be necessary any longer), thesecollections of synthetic biology building blocks will stillbe required for some time, in particular for addressingbasic biological questions and engineering simple phe-notypes in non-E. coli bacteria.

OUTLOOKAs discussed above, environmental plasmids offer syn-thetic biology a diverse palette of gene-encoded activitiesthat once excised from its natural context, minimized,and standardized, become an extraordinary asset for awide range of applications. But there is surely more thatplasmid biology can deliver beyond providing buildingblocks for genetic constructs. First, as mentioned earlier,plasmids, especially those that are promiscuous, movethrough the microbial population, and their encodedgenes are to be expressed in different types of hosts.What determines such ability? Both the plasmid-bornepromoters involved, their regulation, and the proteinsthey encode must operate in a fashion that is relativelyindependent of the host, thereby creating a natural caseof implant-chassis orthogonality that must be ruledby principles that deserve further research. Second, theacquisition of environmental plasmids encoding new

Figure 4 The Standard European Vector Architecture (SEVA) pipeline. (A) The organiza-tion of all SEVA constructs is framed within three basic DNA parts of reference, i.e., twotranscriptional terminators and one oriT. (B) The positions of these parts leave threeopenings flanked by specific and unusual restriction sites which are then used to insertsynthetic DNA fragments encoding an antibiotic resistance marker, an origin of replication,and a cargo segment. The orientation and specifications that such segments must follow toacquire the SEVA standard are described at http://seva.cnb.csic.es. (C) Segments are assem-bled on the formatted frame, and (D) a pSEVA vector is added to the database and to thematerial repository. doi:10.1128/microbiolspec.PLAS-0033-2014.f4

644 PLASMIDS AS GENETIC TOOLS

metabolic capabilities is merely a natural case of bio-chemical engineering where the preexisting molecularnetwork of the receiving cells has to cope with implanta-tion of a new genetic and enzymatic system (97). Studiesof such scenarios (e.g., the interplay of the pWW0-encoded system for toluene and m-xylene degradationand the P. putida host) reveal both the type of biochem-ical conflicts that such situations create and how bacte-ria have found a solution to them (130, 131). Moreinstances of this sort could be worth examining for thesake of handling the problem of implant-chassis retro-activity, one of the central challenges in biological engi-neering (132, 133).

Finally, once plasmids make it to a new host, theirencoded proteins have to be placed somewhere in thetridimensional architecture of the receiving cells, wheremolecular crowding imposes considerable constraints.Are plasmid-encoded functions, cognate protein struc-tures, and processes associated with such three-dimensional addresses? It will be important to classifyplasmid-encoded proteins into those that distributeevenly versus those that go to a specific cell site and tosee what the difference is, thereby understanding theprinciples that may rule such a positioning. In thissense, promiscuous environmental plasmids will con-tinue enriching the material toolbox for engineeringbiological systems as well as providing new conceptualinsights on how to program microorganisms to do(new) things that they normally do not do.

Acknowledgments. The work in the authors’ laboratory wassupported by the BIO program of the Spanish Ministry ofEconomy and Competitiveness, the ST-FLOW, ARISYS, andEVOPROG contracts of the EU, the ERANET-IB Program,and funding from the Autonomous Community of Madrid(PROMPT). Conflicts of interest: We declare no conflicts.

Citation. Martınez-Garcıa E, Benedetti I, Hueso A, deLorenzo V. 2015. Mining environmental plasmids for syn-thetic biology parts and devices. Microbiol Spectrum 3(1):PLAS-0033-2014.

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