1 Synthetic Biology: Implications and Uses COPYRIGHTED MATERIAL€¦ · COPYRIGHTED MATERIAL. 2 Synthetic Biology: Implications and Uses Keywords Synthetic biology An effort to construct

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1Synthetic Biology:Implications and Uses*

Sanjay Vashee, Mikkel A. Algire, Michael G. Montague, and Michele S. GarfinkelJ. Craig Venter Institute, Synthetic Biology, 9704 Medical Center Drive, Rockville,MD 20850, USA

1 Introduction 2

2 DNA Assembly and Modification 5

3 Modular Parts and Circuits 15

4 Spatial Regulation 184.1 Co-Localization 194.2 Compartmentalization 20

5 The Synthetic Cell 21

6 Societal Challenges Posed by Synthetic Biology 23

7 Concluding Remarks 25

Acknowledgments 25

References 26

* This chapter has previously been published in: Meyers, R.A. (Ed.) Systems Biology, 2012,978-3-527-32607-5.

Synthetic Biology: Advances in Molecular Biology and MedicineFirst Edition. Edited by Robert A. Meyers.© 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA.

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2 Synthetic Biology: Implications and Uses

Keywords

Synthetic biologyAn effort to construct biological systems, which may include entire biosyntheticpathways, synthetic organelles and cellular structures, and whole organisms, thathave medical, industrial, and scientific applications. This is achieved via the applicationof engineering principles, such as hierarchical design, modular reusable parts, theisolation of unrelated functions, and standard interfaces.

Synthetic cellA cell that is controlled solely by a genome that was assembled from chemicallysynthesized pieces of DNA.

DNA assemblyThe building of larger DNA fragments from smaller DNA fragments.

CircuitsA collection of various modular component parts that responds to an input signal thatis then relayed to produce an output signal.

CompartmentalizationThe spatial sequestering of substrates, intermediates, products, enzymes, and activities.

Synthetic biology is an effort to construct and engineer biological systems, rangingfrom individual genetic elements, to biosynthetic pathways, to whole organisms.The results of these engineering efforts can be of great value to human interests suchas medicine and industry. In this chapter, advances in DNA assembly technologiesare reviewed, and how these advanced DNA assembly technologies, in conjunctionwith the application of engineering principles such as modular parts, have facilitatedthe rational engineering of organisms to obtain desired functions or to understandcomplex cellular behavior, are highlighted. The recent creation of a synthetic cellis also described. Finally, the societal concerns posed by synthetic biology arediscussed.

1Introduction

The field of Synthetic Biology can be con-sidered more as an engineering discipline,and less as an empirical science. Effortsto create artificial life systems, both in

biochemical systems [1] and in softwareenvironments [2], may also be consid-ered as Synthetic Biology, though theseare beyond the scope of this chapter.Synthetic Biology is viewed as the effortto construct and engineer biological sys-tems of value to human interests. Such

Synthetic Biology: Implications and Uses 3

efforts can range in scopes far largerthan the traditional genetic engineering ofgenes, to include the engineering of entirebiosynthetic pathways complete with theregulation of the genes in that pathway[3, 4], synthetic organelles and cellularstructures [5], whole organisms [6–9], andeven ecosystems [10–13]. Synthetic Biol-ogy has the ambition to apply classicalengineering principles such as hierarchi-cal design, modular reusable parts, theisolation of unrelated functions, and stan-dard interfaces. The empirical fields thatcorrelate to Synthetic Biology are SystemsBiology, Genetics, and Molecular Biology.

Synthetic Biology is not a new field, butrather extends back into prehistory. Forexample, it has been determined that theprocess of engineering maize – a highlyoptimized domestic agricultural crop plant– from the wild grass teosinte beganover 9000 years ago [14]. The methodused by the pre-Columbian cultivatorsof teosinte was simple artificial selectionwhich, as such, is very slow. However,with the discovery of laws of inheritanceand natural selection [15–17], and thesuggestion that DNA was the chemicalmedium of inheritance [18], the scenewas set to engineer a living system ina far more direct and rapid manner.A prominent example of this is theDupont Escherichia coli strain used for theproduction of 1,3 propanediol, in whichcase an entire biosynthetic pathway hasbeen added to E. coli, and the metabolismof the bacterium substantially alteredto allow for a majority of the carbonfeedstock (glycerol) to be converted intothe economically valuable chemical 1,3propanediol [6, 8, 9]. This feat, whichwas begun prior to the development ofmost of the Synthetic Biology techniquesreviewed in this chapter, took many yearsand substantial investment to achieve. Yet,

with recent advances in the field, suchbioengineering projects will become fasterto develop, easier to operate, and also muchmore ambitious.

During recent years, Synthetic Biologyhas progressed in a manner which is verydifferent from those of other engineeringdisciplines. This is because, unlike archi-tecture or software engineering, there isalready a reservoir of highly sophisticatedand complex functional parts to be foundin Nature, and consequently most effortsin Synthetic Biology have been focused onharnessing that natural resource base. Ingeneral, two basic approaches have beenundertaken to achieve this feat. The firstapproach has been to engineer naturalorganisms so as to incorporate recombi-nant pathways and other such desirableattributes. This method has the advantageof not requiring the capability to build –nor require an understanding of – mas-sive biological systems such as genomesand metabolisms. However, it does havethe disadvantage of being undefined; thatis, whilst certain genes of the organismmight be the result of human intervention,most of the genome remains wild-type,and is neither subject to human controlnor necessarily operating within the limitsof human knowledge.

The alternate approach is to use func-tional components, originally ‘‘mined’’from Nature, such as promoters, ap-tamers, protein–protein interaction do-mains, terminators, or ribosome-bindingsites. These functional components can becataloged and then used to compose largerdefined constructions of genes, pathways,and even whole genomes. Because allthe components of a synthetic biologicalsystem that are constructed in such anapproach have precisely defined proper-ties, a high degree of predictive controlover the final product is afforded. Indeed,


this more defined approach has becomesynonymous with advances in SyntheticBiology.

Historically, one of the main limitationsin following this defined approach relatesto the knowledge of these natural systemsthat serve as a source of parts. As biologyhas been characterized, both new com-ponents for synthetic biology – and alsonew tools to utilize those components –have become available. In turn, a newSynthetic Biology capability has drivengreater advances in the understanding ofbiology. This has been most obvious inthe development of tools for the synthe-sis and manipulation of DNA, the first ofwhich were developed via the discoveryof restriction endonucleases, DNA ligases,and the creation of recombinant DNAmolecules [19–24]. These tools allowedthe development of the recombinant DNAcloning and expression techniques that ul-timately made possible the exploitation ofenzymes with desired activities and prop-erties. Notably, the development of thepolymerase chain reaction (PCR) greatlyincreased the ability to amplify and ma-nipulate DNA [25–27]. The subsequent

combination of recombinant DNA cloningtechniques with the PCR allowed a muchgreater exploitation of natural biologicalcomponents, such as thermostable DNApolymerases, and this in turn made thePCR more robust and practical. Today, thePCR has become an indispensable tool forbiology. Thus, knowledge of natural sys-tems has led to an improved technologyfor exploiting those systems, which hasin turn provided an improved knowledgeof biological systems in a double feed-back loop, thus improving both scientificunderstanding and technological capabili-ties. As shown in Fig. 1, as knowledge ofthe fundamental principles of biology havecontinued to grow, it has been possible totake a more defined engineering approach.

In the past, advances in synthetic biol-ogy have been bounded by the capacityto assemble and modify DNA, as wellas knowledge of biological parts and cir-cuits that such DNA might encode. Cor-respondingly, these two areas are directlyaddressed in the following sections, withdetails of synthetic biological pathways,synthetic genomes, synthetic organelles,and even synthetic organisms provided as

Progress in Synthetic Biology is defined by the shifting of life-manipulation from the undefined to defined products and techniques.

WhollyDefinedCustomBiologicalSystems

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(fast, powerful, butrequires substantialbiological knowledge)

(slow, limited in scope,but requires little or nobiological knowledge)

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Cross Hybridization +Artificial Selection

Natural Variation +Artificial Selection

Fig. 1 Increasingly defined biological engineering. Aschematic of how biological engineering has emphasized amore defined and rational design as it has advanced, basedon a greater knowledge of natural biological systems.


examples of the capabilities that SyntheticBiology has already begun to deliver.These new capabilities create, in turn,new societal challenges, and these are alsodiscussed.

2DNA Assembly and Modification

As discussed above, one way to viewSynthetic Biology is as an engineeringdiscipline aimed at manipulating cellu-lar systems to produce a de novo-designedfunction that does not exist in the nat-ural organism. As with other engineer-ing fields, however, Synthetic Biology isdependent on the tools and techniquesavailable.

Organisms carry out a variety of reac-tions aimed at their self-sustenance andself-replication, with such reactions beingcarried out by the proteins and RNAs en-coded in the organism’s genome. As thesequence of the DNA directs productionof the proteins and RNAs of an organism,control of the cellular DNA therefore al-lows for an ability to direct the functionsof a cell. Based on this principle, manyof the basic tools utilized in SyntheticBiology are aimed at producing definedsequences of DNA molecules, and eas-ily manipulating the DNA content of anorganism. The DNA molecules necessaryfor Synthetic Biology purposes can varygreatly in length, from individual DNAparts, genes and plasmids (containing tensto thousands of base pairs) to biosyntheticpathways and genetic circuits (thousandsto millions of base pairs) to synthesizingwhole genomes (viral and bacterial).

For many years, gene cloning and DNAassembly were dominated by the useof restriction endonucleases and DNAligases [19–24]. While some well-designed

restriction enzyme-based methods are stillcommonly in use [28, 29], these methodsare gradually being superseded by the de-velopment of very rapid, more robust andless limited DNA assembly techniques.For example, although BioBricks wereoriginally designed to be assembled witha restriction enzyme/ligation method [30],more recently an in vitro homologousrecombination was adapted to increasethe flexibility and speed of BioBrickassembly [31].

The starting materials for DNA se-quence construction may include chem-ically synthesized DNA oligonucleotides(oligos), natural DNA fragments, PCRproducts, or a combination of all threesources. Defined short single-stranded oli-gos have been commercially available as acommodity for many years, usually for useas primers in PCR, mutagenesis, and se-quencing reactions. Since chemically syn-thesized DNA oligos are of a user-definedsequence, this allows for a nucleotide levelcontrol of gene sequences and even entiregenome sequences – a firm requirementwhen designing new functions in organ-isms. The idea of synthesizing genes fromDNA oligos is not new; previously, oligoshave been used to synthesize genes suchas the alanine tRNA from yeast [32] andthe human leukocyte interferon gene [33].Likewise, the gene encoding a mammalianhormone, somatostatin, was synthesizedand expressed in E. coli [34]. It is onlyrecently that the lower costs of oligonu-cleotide synthesis and DNA sequencinghave been combined to allow the devel-opment of more cost-effective and rapidmethods for assembling groups of oligosinto synthetic pieces of DNA or genes[35–39]. Today, several commercial genesynthesis companies exist that are able toproduce custom genes/DNA at an accessi-ble cost per base pair, although such costs


can quickly become prohibitive if numer-ous different DNAs are required. The costprohibition of large-scale gene synthesiscan, in part, be overcome by utilizing nat-ural DNA fragments and PCR productsin the DNA assembly for those sectionsof DNA that do not need to be createdsynthetically.

Typically, the process of constructingDNA is hierarchical (Figs 2 and 3).Briefly, groups of smaller DNAs (single- ordouble-stranded) are mixed and assembledinto larger DNA pieces. Figure 2 showsdouble-stranded DNAs being assembledinto a larger construct, but the process canbegin with single-stranded oligos as thesubstrates. These larger pieces (subassem-blies) are then grouped and assembled.These steps can be repeated until the fi-nal full-length DNA construct is obtained,whether it is a gene or genome. The DNApieces to be assembled must have homol-ogous overlapping ends, the overlaps be-ing important because the DNA assemblytechniques utilize homologous recombi-nation. For example, if three pieces of DNA(A, B, and C) are to be assembled into a sin-gle DNA molecule, then one end of piece Amust have an overlap with piece B, and theother end of B must overlap piece C. Thisconfiguration will result in a linear DNAmolecule, A–B–C. In order to generate acircle from these pieces, the end of C mustoverlap piece A. The assembly of DNAinto a circle is most often achieved witha DNA piece which contains sequencesthat enable the final construct to be clonedinto a desired host (e.g., E. coli, Saccha-romyces cerevisiae or Bacillus subtilis) [37, 38,40–42]. The DNA homologous recombina-tion reaction can be carried out completelyin vitro in one step, either with an enzymemix [37] or by using the PCR [35]. Thereaction can also be performed in vivo by

utilizing the natural homologous recom-bination activity of an organism, such as S.cerevisiae (yeast) and B. subtilis [7, 40–44].Occasionally, a method will include anin vitro step to perform a partial reaction(DNA chewback/DNA annealing/DNA ex-tension), and an in vivo step to completethe reaction (DNA repair) [38, 45, 46].

The various in vitro homologous re-combination methods used to assembledouble-stranded DNA or single-strandedoligos share the same general mechanism(Fig. 2). The nucleotides are first removedfrom one strand of the overlapping ends ofthe adjacent double-stranded DNAs, thuscreating single-stranded ends of the DNA(Step 1). This process is analogous to arestriction enzyme digestion creating com-plementary sticky ends of DNA, exceptthat the single stranded ends are typically20–60 nucleotides long. Depending on themethod used, the nucleotides can be re-moved by applying an exonuclease activityfrom either the 5′ or 3′ ends of the DNA[37, 38]. The creation of single-strandedoverhangs (Step 1) is not necessary if oli-gos are used as the starting material forDNA assembly, because the oligos are al-ready single-stranded. The single-strandedDNA overhangs are complementary toeach other on adjacent molecules, and thusare able to anneal (Step 2). If the ends ofthe molecule being constructed are com-plementary, then the final construct willbe circular (this is the normal methodused when DNA is being assembled intoa cloning or expression vector). The finalstep to the reaction is repair of the DNA.In the in vitro reaction, a DNA polymeraseis used to fill the gaps, while a DNA ligaseseals the nicks so as to create the larger as-sembled DNA molecule. The DNA repairactivity of E. coli can be utilized to com-plete the reaction after the annealing step


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Fig. 2 Schematic depicting in vitro homol-ogous recombination DNA assembly. First,nucleotides are removed from either the 5′ orthe 3′ ends of the DNA pieces (5′ removal de-picted). This step can be performed by severalenzymes. The newly exposed single-strandedhomologous ends (red, green, or yellow re-gions) on the adjacent pieces are comple-mentary, and can anneal. Providing homologyat the ends of the DNA pieces will result inthe assembly of a circular DNA molecule.

Following the annealing step, the DNA is re-paired by filling in the gaps with a DNA poly-merase and sealing any nicks with DNA ligase.A linear assembly product can be amplified byusing the PCR and used in further assemblyreactions. The circular assembly products aretransformed into the appropriate host in or-der to isolated individual clones with the finalassembled molecule. The assemblies can alsobe repaired in vivo after transformation by thenative activities of E. coli.


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[38] and after other enzymatic steps thatremove errors [36]. The final assembly canbe amplified by using the PCR and, if nec-essary, used in another round of assemblyto generate even larger DNA molecules.If the final assembly is circular. it canbe transformed into the appropriate hostin order to generate individual assemblyclones.

The construction of DNA from oligoscan also be performed in vivo using the re-combination activity of an organism. Theyeast S. cerevisiae has robust homologousrecombination activity, as well as an abil-ity to take up multiple pieces of double- orsingle stranded DNA [47–51]. Previously,Gibson and colleagues have shown thatyeast can assemble at least 38 overlappingsingle-stranded 60-mer oligos directly intoa plasmid, thus forming a 1170 bp DNAinsert. Alternatively, fewer – but longer –oligonucleotides (up to 200 nt in length)that overlap by as little as 20 bp can alsobe used to assemble 1100 bp assembliesdirectly into the desired plasmid [43]. Theability of yeast to support at least 2 Mb ofcloned DNA also makes yeast a good hostfor assembling large constructs.

At this point, a discussion of genesynthesis and DNA assembly methods,within the context of the synthesis of threedifferent genomes, will be used to high-light and demonstrate a variety of DNAassembly methods that have been used tosynthesize DNA sequences, starting fromsingle-stranded oligos, and the hierarchi-cal assembly of the DNA subfragmentsinto complete genomes. This is not meantto be a comprehensive list of all avail-able methods and techniques; rather, theintention is to demonstrate the flexibilityof recently used methods in DNA assem-bly. Whilst the discussion will be withinthe framework of whole genome synthe-sis, these techniques can also be used tosynthesize and/or assemble any DNA ofinterest, from a few base pairs in length toover a million.

Gibson and colleagues have synthesizedthree genomes using both in vitro andin vivo assembly techniques [7, 40, 41].Figure 3 shows a schematic flow ofthe hierarchical synthesis of the mousemitochondrial genome (16 299 bp) (thishas also been assembled, using a differentmethod, by Itaya et al.; see below),

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Fig. 3 Synthesis of mitochondrial and bac-terial genomes. The hierarchical assembly ofthree genomes is depicted with the sizes ofthe intermediate subassemblies and final prod-ucts on a logarithmic scale. The red arrowsrepresent in vitro assembly, and the greenarrows in vivo assembly in yeast. Values inparentheses indicate the number of piecesat that stage in the assembly. The coloredbars on the left represent the several differentDNA molecule classes that can be produced,and their relative sizes. (a) The mouse mito-chondrial genome was synthesized startingfrom 60 nucleotide-long oligonucleotides infour stages. All of the assembly steps wereperformed in vitro; (b) The Mycoplasma gen-italium genome was assembled from 5 to7 kb cassettes purchased from a custom DNA

synthesis company. Both, in vitro and in vivoDNA assembly techniques were utilized in thegenome construction. The final stage of theassembly was performed in vivo, using yeast.The ability of yeast to take up multiple DNApieces can eliminate several rounds of con-struction, as demonstrated by the 25-piecein vivo assembly of the genome; (c) The My-coplasma mycoides ssp. capri genome wasassembled from over one thousand 1080 bppurchased DNA cassettes. The three assem-bly steps for this genome were all performedin vivo, with yeast as the host. The dottedred and green lines represent the availabilityof several in vitro and in vivo DNA synthesismethods that can be applied to produce theDNA cassettes for use in hierarchal DNA as-semblies.


the Mycoplasma genitalium genome(582 970 bp), and the Mycoplasma mycoidesssp. capri genome (1 077 947 bp). Thebasis of these DNA assembly methods isthe DNA homologous recombination ofoverlapping DNA fragments.

The mouse mitochondrial genome wassynthesized starting from 600 overlap-ping 60 nt-long single-stranded DNAs(60-mers), using an in vitro one-step as-sembly method (as discussed above) andPCR [52]. The first stage of genome con-struction produced 284 bp subassembliesby assembling groups of eight oligos di-rectly into pUC19 (Fig. 3a). Assemblingthe oligos directly into a cloning vec-tor allowed the individual assemblies tobe cloned, isolated, and sequence-verifiedbefore continuing with the constructionprocess. Following sequence verification,the correct 284 bp first stage assemblieswere amplified by PCR in order to generatemore material. Following amplification,the first-stage assemblies were pooled intooverlapping groups of five, and again as-sembled in vitro to produce the 1.2 kbsecond stage assembly intermediates. Thesecond stage assemblies were not propa-gated in a host organism, but rather werePCR-amplified immediately following thein vitro assembly reaction. These 15 PCRproducts were pooled into groups of fiveand then joined to form the 5.6 kb thirdstage assembly intermediates. The assem-bly products were amplified by PCR (asbefore), and these three PCR productswere then assembled to form the completesynthetic mouse mitochondrial genome.The final assembly reaction with the threePCR products also contained a bacterialartificial chromosome (BAC), so that thefinished mitochondrial genome could becloned into E. coli. This case demonstratesnot only the ability to perform large-scaleDNA construction almost entirely in vitro,

but also that the process is amenable toautomation.

In 2008, the first synthetic bacterialgenome was synthesized at the J. CraigVenter Institute [40]. The process to assem-ble the synthetic 582 970 bp Mycoplasmagenitalium genome was hierarchical – sim-ilar to the mouse mitochondrial genomeassembly (although the M. genitaliumgenome is about 35-fold larger). The syn-thetic M. genitalium genome was assem-bled from 101 synthetic DNA cassetteseach of about 5–7 kb in length (Fig. 3b).In this case, the cassettes were synthe-sized from oligos by several different genesynthesis companies, and verified by se-quencing. The cassettes overlapped theiradjacent neighbors by an average of about80 bp. In order to allow the formation ofthe circular genome, cassette 1 overlappedcassette 101.

The main challenge in the synthe-sis of the M. genitalium genome wasthe assembly and cloning of syntheticDNA molecules larger than those previ-ously known. In the first stage, sets offour adjacent cassettes were assembled byin vitro recombination into a BAC vector toform circularized recombinant plasmidswith about 24 kb inserts that were thenreleased by restriction enzyme-mediateddigestion in preparation for the next as-sembly stage. The 25 first-stage assem-blies were taken three at a time to formthe 72 kb second-stage assemblies, againby in vitro recombination. In the thirdstage, the 72 kb second-stage assemblieswere taken two at a time to producefour third-stage assemblies, each of ap-proximately one-quarter-genome (144 kb)in size. The first three stages of assemblywere performed by in vitro recombina-tion and cloned into E. coli in order togenerate more DNA for the subsequentrounds of assembly. The final stage of


the M. genitalium genome assembly wascarried out in vivo by utilizing the homolo-gous recombination activity of S. cerevisiae.The last stage of the genome assembly con-sisted of six overlapping pieces of DNAto generate the complete M. genitaliumgenome (one yeast vector, two fragmentsof quarter 3, and quarters 1, 2, and 4).The final step was performed in vivo be-cause limitations of the cloning host andin vitro assembly reaction became appar-ent. It is possible that larger assemblies(280–580 kb) are not stable in E. coli, butit is also possible that the circularizationof large DNA molecules may be inefficientduring the in vitro recombination reaction,and/or that handling large DNA moleculesin solution leads to breakage of the DNAbefore transformation.

Subsequently, the powerful ability ofyeast to take up and assemble multiplelarge fragments of DNA was demonstratedby taking the 25 first-stage assemblies andassembling them in one step by usingyeast [41]. This proved to be significantbecause it allows for fewer assembly steps,and thus greatly reduces the time requiredto construct large DNAs.

By leveraging the DNA uptake andrecombination capability of yeast, athree-stage hierarchical strategy wasdesigned to assemble the 1 077 947 bpM. mycoides ssp. capri genome. In thiscase, the assembly steps were performedentirely in vivo by transformation andhomologous recombination in yeast,following the initial DNA cassette con-structions (Figs 3c and 4) [7]. This differsfrom the strategy used to construct themouse mitochondrial and M. genitaliumgenomes, which used in part an in vitrohomologous recombination reaction. Thecassettes designed to assemble into thecomplete genome were generally each of1080 bp, with 80 bp overlaps to adjacent

cassettes. As with M. genitalium, the 1078cassettes (each 1080 bp long) were allproduced commercially by the assemblyof chemically synthesized oligos. Toassist in the assembly process, DNAcassettes and assembly intermediateswere designed to recombine in thepresence of vector elements to allow forgrowth and selection in yeast. Duringthe first stage of assembly, groups of10 of the 1080 bp DNA cassettes anda vector were recombined in yeast toproduce circular subassembly plasmids;these were then transferred to E. coli inorder to easily generate the quantitiesof subassembly DNA required for thesecond-stage assembly step.

For the second-stage assemblies, 10 ofthe 10 kb assemblies were pooled and theirrespective cloning vectors transformedinto yeast to produce 100 kb assemblyintermediates. Circular plasmid DNA wasextracted from yeast in order to proceedto the final assembly stage. In the finalstage, 11 of the second-stage assemblies(100 kb each) were pooled, and the yeasttransformation procedure was repeateda final time to produce the circular M.mycoides ssp. capri genome.

Recently, various alternatives to us-ing yeast to clone and assemble largeDNAs in vivo have been described. Forexample, Itaya and colleagues also con-structed the complete recombinant mousemitochondrion (16.3 kb) and rice chloro-plast (134.5 kb) genomes from their smallcontiguous DNA pieces. In these cases,the starting DNAs for the assembly werederived via a PCR and assembled in B. sub-tilis [53]. The latter bacterium has a verylarge capacity to uptake and assembleDNA, as demonstrated by cloning of the3.5 Mb genome of the photosynthetic bac-terium Synechocystis into the B. subtilisgenome [42, 54]. This was accomplished


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Fig. 4 The assembly of a synthetic M. my-coides ssp. capri genome in yeast. A syntheticM. mycoides genome was assembled from1078 overlapping DNA cassettes in threesteps. In the first step, 1080-bp cassettes (or-ange arrows), produced from overlapping syn-thetic oligonucleotides, were recombined insets of 10 to produce 109 approximately 10 kbassemblies (blue arrows). These were thenrecombined in sets of 10 to produce 11 ap-proximately 100 kb assemblies (green arrows).In the final stage of assembly, these 11 frag-ments were recombined into the completegenome (red circle). With the exception of

two constructs that were enzymatically piecedtogether in vitro (white arrows), assemblieswere carried out by in vivo homologous recom-bination in yeast. Major variations from thenatural genome are shown as yellow circles.These include four watermarked regions (WM1to WM4), a 4 kb region that was intention-ally deleted (94D), and elements for growthin yeast and genome transplantation. In ad-dition, there are 20 locations with nucleotidepolymorphisms (asterisks). Coordinates of thegenome are relative to the first nucleotide ofthe natural M. mycoides ssp. capri sequence.The designed sequence is 1 077 947 bp.


by progressively assembling and editingcontiguous DNA regions that cover theentire Synechocystis genome. It is impor-tant to note that the Synechocystis genomewas not a circular free molecule (as arethe other genomes), but rather was incor-porated as two pieces into the B. subtilisgenome.

Several general features have been iden-tified that might be desirable in a DNAsynthesis method. Although, ideally, themethod should have a low cost per basepair of DNA synthesized, the present costof DNA synthesis is dominated by the priceof the starting oligos. Oligonucleotides ob-tained from DNA microarrays can greatlyreduce the cost of gene synthesis (esti-mated to be as much as an order ofmagnitude), because thousands of oligoscan be synthesized on a single chip ona small scale. Gene synthesis from DNAmicroarrays can be hampered by the smallquantity and complex mixture of the oli-gos obtained [55–57]. Regardless of thesource of oligos, the cost of DNA synthe-sis can be greatly impacted by the amountof sequencing required to find a correctclone, and this leads to the importanceof accuracy in gene synthesis. Errors ingene synthesis are unavoidable becausethe process of creating the starting DNAoligos is not perfect, and some fractionof the oligos will inevitably contain errors.Whilst the starting oligos can be purifiedby using different methods in order tominimize the number of error-containingoligos, such processes are expensive andtime- consuming, and also are not com-patible with high-throughput gene synthe-sis. The sequencing of multiple clones issometimes sufficient to identify the cor-rect synthesized DNA, depending on theefficiency of the assembly method usedand the size of the DNA construct. Sev-eral error-correction methods have been

developed, however, in attempts to reducethe amount of sequencing required [36,58, 59].

The goals of Synthetic Biology often re-quire DNA to be manipulated from thenucleotide to the genome level. Althoughthe methods available to generate syntheticDNAs from genes to genomes have beendiscussed, in many cases a genome mayneed to be modified by inserting, deleting,or replacing a gene or sequence. Thesemodifications may be necessary in only afew places, either individually or simulta-neously in several noncontiguous places.Consequently, several DNA manipulationtechniques have been developed to allowthese types of change.

Recombineering uses the activity oflambda phage enzymes to catalyze highlyefficient homologous recombination invivo [60]. The lambda Red system allows aneasy and rapid modification of the genomeof a compatible host. However, becauseonly short homologous overlaps are re-quired for recombination with the lambdaRed system, it is possible to use PCR toeasily generate the modifying DNA by in-corporating the homologous overlaps intothe primer design. Recombineering canbe used to insert, delete or replace a geneor sequence. Moreover, if multiple modi-fications are needed in the same organismthe process can be repeated sequentially,although the time required to make morethan a few changes can become signifi-cant. This system is available for E. coli [61,62], Pseudomonas [63], and Salmonella [64],while a similar system based on a differentphage has been developed for Mycobac-terium tuberculosis and M. smegmatis [65].Based on the studies with M. tuberculosis,it is not unreasonable to speculate thata similar recombineering system can beimplemented in many more organismsby exploiting their native phages.


By using the same lambda recombi-nation proteins, Wang and colleaguesdeveloped the technique of multiplex au-tomated genome engineering (MAGE) forthe large-scale programming and evolu-tion of E. coli cells [66]. MAGE employs amixture of single-stranded oligos to simul-taneously target many locations on thechromosome for modification either ina single cell, or across a population ofcells. Selection markers are not necessarybecause the process is efficient, iterative,and cumulative. The highest efficiencies ofMAGE are observed when small changesare being made to the genome (a fewbase pairs), but the efficiency is muchreduced when larger changes such as in-sertions (>20 bp) or deletions (>1000 bp)are attempted. The MAGE process is ablerapidly to produce combinatorial genomicdiversity. Indeed, the power of MAGEhas been demonstrated by tuning thetranslation of 20 endogenous genes andoptimizing the production of lycopene inE. coli. Warner et al. have combined amolecular barcode technology with recom-bineering to develop trackable multiplexrecombineering (TRMR) [67], such thatthousands of specific genetic modifica-tions can be produced simultaneously, byrecombineering. In this case, each mod-ification is associated with a molecularbarcode, and the barcode sequences andmicroarrays can then be used to quan-tify the allele frequency in the population;this, in turn, allows mapping of the geneticmodifications that affect a trait of interest.This technique may be useful when engi-neering a trait for which there is limitedgenetic knowledge.

A method for the rapid engineeringof multiple genetic changes in yeastwas developed by Suzuki and colleagues[68]. This method, referred to as ‘‘GreenMonster,’’ employs an inducible green

fluorescent protein (GFP) reporter geneto create individual deletions in sepa-rate yeast strains. The deletions in theindividual strains are then combinedby repeated rounds of mating, meiosis,and flow cytometry-based enrichment. Ineach sexual cycle, progeny bearing anever-increasing number of altered loci areenriched on the basis of gene dosage.If it could be adapted to extrachromo-somal DNAs, the Green Monster mightrepresent a valuable technique for al-tering large DNAs or bacterial genomeswhich are cloned in S. cerevisiae. Althoughthe method has been demonstrated usingS. cerevisiae, it should be possible to extendthe technology to bacteria with a matingequivalent such as bacterial conjugation(e.g., E. coli).

In some cases, when the expressionfrom a high gene dosage is desired, the useof plasmids may not be a feasible option.In this case, copies of the gene of interestcould be inserted into the genome by usingsome of the above-described methods, butthis may be both labor- and time-intensiveif a high gene dosage is required. In an at-tempt to overcome this problem, Tyo andcolleagues used a plasmid-free, high-genecopy expression system termed chemicallyinducible chromosomal evolution (CIChE)to evolve an E. coli chromosome with about40 copies of a recombinant pathway [69].This was achieved by creating a cassettethat contained the genes of interest, alongwith a gene encoding antibiotic resistancefor chloramphenicol. When the strain wasthen grown in increasing concentrationsof chloramphenicol, the selective pressureof the increasing antibiotic concentrationresulted in duplications of the cassette and,therefore, also of the antibiotic resistancegene by recA-dependent homologous re-combination. When the desired cassettecopy number was reached, recA could


be deleted to prevent any homologousrecombination that could alter the cassettecopy number.

As shown above, many good methodshave been devised for constructing and as-sembling either synthetic or natural DNA.However, several factors must be consid-ered and balanced when deciding on aDNA assembly strategy; these should in-clude the cost of the method, the timerequired, the individual’s experience withthe different host organisms, and whetherthere is a need for vast combinatorial num-bers. Fortunately, the different assemblymethods are diverse and robust, so that alarge range of requirements can be met.In general, it appears that yeast may bethe preferred host when constructing largeDNAs, although several of the methodsavailable to generate genes or smaller (ca.1 kb) subassemblies for the constructionof larger DNAs are equally suitable, de-pending on the user’s preferences.

3Modular Parts and Circuits

One core component of Synthetic Biologyis to apply engineering-based approachesof modularization, rationalization, andmodeling to control cellular behavior, inorder to obtain desired functions or tounderstand complex biological systems[70, 71]. As a result, an increasing numberof synthetic biologists have begun to applyelectrical circuit analogies to biologicalpathways as a means of designing andgenerating synthetic genetic devices whichcan then be placed into cells to controltheir behavior [72]. The first examples ofgenetic circuits – the toggle switch and therepressilator – were demonstrated about adecade ago [73, 74], since when there hasbeen an ever-expanding number of reportsof various types of biological circuit,

including additional genetic switches[75, 76], other oscillators [77–80] andmemory networks [81, 82], as well as otherelectronic-inspired genetic devices [70]such as pulse generators [83], logic gates[75, 84], filters [85], and communicationsmodules [86, 87]. Today, these deviceshave begun to be used for practicalapplications in biosensing, therapeutics,and in the generation of importantindustrial products. As the scope of thischapter is very broad, it is only possiblehere to discuss these synthetic geneticdevices and their uses with limitedrepresentative examples. A number ofexcellent recent reviews will provide moredetailed information on these syntheticgene networks [70, 71, 88–90].

The basic design of circuits is the assem-bly of various modular component partsthat respond to an input signal that is thenrelayed to produce an output signal. Forbiological circuits, the component partshave their origins in the vast amount ofbasic research in all aspects of the biologi-cal functions of organisms. The combinedeffort of many research groups has led togreat understanding of the various path-ways that organisms use to respond toenvironmental signals, such as light orquorum sensing. Accordingly, many of thecomponents of these pathways – such assignaling proteins and transcription fac-tors, as well as the promoters they control– have been identified from a wide va-riety of organisms [71, 89, 91]. In orderto create some biological devices, syn-thetic biologists have taken componentsfrom one organism and placed them ina different model organism. For example,Danino et al. synthesized synchronizedgenetic oscillators by placing elementsof the quorum sensing machineries ofVibrio fischeri and Bacillus thurigensis intothe model organism, E. coli [77]. Given


the difference in GC (guanine–cytosine)content, codon usage, transcription andtranslation processes between the vari-ous organisms, it was not initially clearwhether the devices created from the dif-ferent organism-derived component partswould function as intended. However, toovercome this difficulty several methodshave been adapted.

First, web applications such asGeneDesign [92] have been developedthat enable synthetic biologists to designthe component parts according to thespecifications of the host organism(see Table 1). In addition, as discussedabove, the recent advances in genesynthesis, as well as the decrease in thecost of oligos and the proliferation ofgene synthesis companies, have allowedsynthetic biologists simply to purchasethe component parts.

Second, Knight, Rettberg, Endy and oth-ers have founded the Registry of StandardParts (Table 1) to provide a framework forsynthetic biologists to devise biological cir-cuits, pathways, and other genetically en-coded systems. The idea here is to emulatethe engineering principles involved in theconstruction of such things as electronicdevices [93]. The Registry of Standard Partsprovides a catalog of a wide variety ofbiological parts, such as transcription pro-moters and terminators, ribosome bindingsites and regulatory proteins, as well as avariety of chassis in which the parts canfunction. This facilitates the ability of thesynthetic biologists to tailor their biolog-ical devices by allowing them to choosefrom a variety of parts whilst, at the sametime, providing a wealth of information onhow these parts can function. The Registryalso allows for interfacing with other webapplications such as modeling tools. Asan example, users can input informationfrom the Registry into SynBioSS designer

to generate kinetic models for the selectedbiological constructs, and provide a pictureof how these constructs would influencethe behavior of the whole [94].

With these advances in place, syntheticbiologists are today beginning to use DNAassembly techniques to piece togethermodular parts into controlled biologicalpathways as well as biological circuits ina wide spectrum of applications, such asbiosensing, the production of therapeu-tics and biofuels, as well as understandingcomplex cellular behavior and even ecosys-tems. For example, synthetic biosensorscan be used to detect various environ-mental signals and then to prompt cellsto enter a programmed behavior [70]. Inone study, Kobayashi et al. generated agenetic device that could detect DNA dam-age and, through a designed activation ofthe SOS pathway, program E. coli cells toenter a biofilm state [95]. The ability wasalso demonstrated of designed bacteria toproduce invasin from Yersinia pseudotuber-culosis, upon the detection of an hypoxicenvironment of tumor cells, which in turnallowed the bacteria to invade the tu-mor cells [96]. In a particularly innovativeexperiment, Looger et al. computation-ally redesigned protein–ligand specifici-ties to construct receptors that could bindtrinitrotoluene or l-lactate. These recep-tors were then incorporated into syntheticbacterial signal transduction pathways toregulate gene expression in response to ex-tracellular trinitrotoluene or l-lactate [97].The results of these studies confirm thepromise that genetic biosensors of thistype may be useful for detecting any de-sired environment or signal, and to allowthe host organism to respond in a targetedmanner.

Biological circuits have also shown theirpossible value in the antibacterial thera-peutic field. For example, bacteriophages


Tab. 1 Resources for synthetic biology.

Resource URL Comments

Synthetic BiologyResources

http://www.istl.org/10-spring/internet1.html

Provides links to various resources on theinternet, including synthetic biologyassociations, centers of research, ethics,training and educational resources, andjournals

SyntheticBiology.net

http://www.syntheticbiology.net/index.aspx

Portal for professionals in syntheticbiology providing information on news,events, products, suppliers, etc.,regarding synthetic biology

Synthetic BiologyProject

http://www.synbioproject.org/ Established as an initiative of theForesight and Governance Program ofWoodrow Wilson International Centerfor Scholars to foster informed publicand policy discourse concerning theadvancement of Synthetic Biology

BioBricksFoundation

http://bbf.openwetware.org/ Encourages the development andresponsible use of technologies basedon BioBrick™ standard DNA parts toallow synthetic biologists to programliving organisms in the same way acomputer scientist can program acomputer (see below)

Registry ofStandardBiological Parts

http://partsregistry.org/Main_Page

A collection of genetic parts that can bemixed and matched to build syntheticbiology devices and systems

SynBERC SyntheticBiologyEngineeringResearch Center

http://www.synberc.org/ Mission is to develop technologies tobuild biological components andassemble them into integrated systemsto perform designed tasks, trainengineers for biology, and educate thepublic on Synthetic Biology

BIOFAB http://www.biofab.org/ Biological design–build facility that aimsto produce useful collections ofstandard biological parts available toacademic and commercial users

JBEI Registry https://public.jbeir.org/ Also aims to provide standard DNA partsfor Synthetic Biology

GeneDesign http://www.genedesign.org/ Set of web applications that providespublic access to a nucleotidemanipulation pipeline for SyntheticBiology

SynBioSS Designer http://synbioss.sourceforge.net/ Software suite for the generation, storage,retrieval, and quantitative simulation ofsynthetic biological networks


were engineered to suppress the SOSpathway of bacteria and enhance thekilling of antibiotic-resistant bacteria,‘‘persister cells,’’ and biofilm cells [98].Another area where the ability to easilydesign and place modular parts into adesigned biological pathway is provinguseful is in the control of metabolic fluxfor the production of industrially impor-tant materials or chemicals. Recently, theStephanopoulos group has reported anability to increase the titer of taxadiene(an intermediate of the potent anticancerdrug, Taxol) in an engineered E. coli strain[99]. This effect was accomplished byfirst partitioning the taxadiene metabolicpathway into two modules – a nativeupstream methylerythritol-phosphate(MEP) pathway forming isopentenylpyrophosphate, and a yew tree-based het-erologous downstream terpenoid-formingpathway – and then varying the module’sexpression simultaneously to obtain animproved balanced pathway.

Genetic devices are also proving theirworth in helping to understand the un-derlying basic principles of coordinatedcomplex cell behavior. As examples, tworelatively recent studies have highlightedefforts to emulate pattern formation that isimportant for development in higher eu-karyotes [85, 100]. The latter study usedan in vitro approach with DNA-coatedparamagnetic beads fixed by magnetsin an artificial chamber to form arti-ficial transcription–translation networksthat generate simple patterns [100]. In theformer study, Basu et al. engineered twogenetically distinct populations of bacte-ria (acyl-homoserine lactone senders andreceivers) and manually overlaid them indifferent configurations to produce differ-ent patterns [85]. More recently, insteadof using two distinct bacterial popula-tions, Tabor et al. genetically engineered an

isogenic community of E. coli cells to senselight, to communicate to identify light-darkedges, and produce an image [101]. Similarbiological circuits to those discussed abovehave also been utilized to construct syn-thetic ecosystems or biofilms to model andbetter understand microbial communities[10–13]. While relatively simple geneticdevices have been used thus far, it is ex-pected that a thorough characterizationof their performance, together with im-proved predictive mathematical tools, willallow for the design and construction ofmore elaborate circuits to program cellsand cellular communities for functionsthat mimic those of natural systems.

4Spatial Regulation

Many of the parts and circuits detailedabove involve the regulation of genes orgene-products. For most of the time, whengene regulation is referred to, it is assumedto be a temporal regulation – that is, theincrease or decrease of expression of agene that was, at a previous time, in theother state. It is, however, also possibleto regulate genes and gene products inspace. The ability to compartmentalize orlocalize enzymatic activities has long beenrecognized as a powerful means of opti-mizing catalysis. In Nature, this can takethe form of macromolecular complexesthat actively channel substrates from oneenzymatic active site of a biosynthetic path-way to the next [102–105], organelles andcompartments that can physically sepa-rate certain enzymes and substrates fromthe rest of the cellular processes [106,107], or it may simply be the result ofreduced diffusion due to co-localization[108, 109]. Just as compartmentaliza-tion and co-localization can take manyforms, so too they offer many advantages.


These include the mitigation of toxicity ofintermediates of a biosynthetic pathway,the protection of intermediates from diffu-sion or degradation, the elimination of un-productive side reactions as a consequenceof the other biological activities of the hostorganism, or improving activity by drivingkinetics (for an excellent review of bothnatural and engineered systems of spatialcontrol over cellular processes, see Ref.[108]). In the past, organic chemists havebeen inspired by these natural systems andhave created a wide variety of biomimeticcatalysts that incorporate such featuresas cyclodextrin covalent linkers that allowmany enzyme molecules to be tethered toone another, leading to improved kinetics[110]. However, whilst inspired by biol-ogy, and often using components derivedfrom living organisms, such systems arenot wholly biological and rarely operatein the biological context from which theircomponent enzymes are derived. Conse-quently, such cell-free systems, which lackthe self-replication capability of living cells,often suffer from problems of enzyme sta-bility and purification. An excellent reviewof biochemical constructs that incorporatecompartmentalization and co-localizationbased around such diverse methods as co-valent and noncovalent linkers, of micellesmade from both synthetic polymers andlipids, and of vesicles and viral particlesused as nanoreactors, has been preparedby Vriezema et al. [111].

Here, examples of engineered, cellularcompartmentalization strategies will bediscussed, with emphasis placed on the de-sign principles that they embody and use.

4.1Co-Localization

Dueber et al. were able to use the syntheticbiology principles of modular parts and

co-localization to create a heterologoussynthetic protein complex in E. coli of threeenzymes: acetoacetyl-CoA thiolase (A toB); hydroxy-methylglutaryl-CoA synthase(HMGS); and hydroxymethylglutaryl-CoAreductase (HMGR) [3]. These threegenes, derived from yeast, compose apathway that produces mevalonate fromacetyl-CoA. Mevalonate is a precursorfor the production of chemicals in theindustrially and medically valuable largeisoprenoid family [112]. However, dueto very different levels of activity, theseenzymes – even if expressed at optimallevels – result in a build-up of the toxicintermediate hydroxymethylglutaryl-CoA(HMG-CoA). To overcome this, Dueberet al. organized the three-enzyme pathwayinto a synthetic complex of enzymes on a‘‘scaffold’’ by using the well-characterizedsignal processing protein–proteininteraction domains of the metazoan cells[mouse SRC Homology 3 (SH3) and PostSynaptic Density protein, Drosophila DiscLarge Tumor Suppressor, and ZonulaOccludens-1 protein (PDZ) domains andthe rat GTPase protein Binding Domain(GBD domain] with their correspondingligands. Each ligand is a small tag-likesequence that can be added to either endof a protein, and which binds specificallyto its corresponding domain. The SH3,PDZ and GBD domains were thenexpressed as a fusion protein that wouldrecruit the three ligand-tagged enzymesof the mevalonate pathway into a singlecomplex or ‘‘scaffold.’’ The main aspectsof the scaffold optimization and designinvolved the order of the binding domains(and, consequently, of the pathwayenzymes with their ligands), and whetherthe ligand sequence was fused to the Nor C terminus of each enzyme. Anotheraspect to be optimized was the numberof each enzyme that was recruited to the


scaffold. Typically, a scaffold might bedesigned to have three PDZ domains, butonly one SH3 and one GBD domain; thiscaused three copies of one of the pathwayenzymes to be recruited to the complex,but only one each of the other enzymes.When these optimizations were made, theresult was a dramatic 77-fold increase inthe yield of mevalonate due to a reductionin the metabolic load on the cell andtoxicity associated with HMG-CoA buildup. The general nature of the methodwas also proved by its application to thepathway for glucaric acid synthesis [3].

The principle of synthetic complexes andco-localization can be used to improve notonly synthesis pathways but also degrada-tive pathways. For example, the fungalcellulase Cel6A has been docked ontoa bacterial mini-cellulosome to achievea greater cellulose degradation and, aswith the mevalonate biosynthetic path-way discussed above, issues of geometryand organization of the final syntheticscaffold strongly affected the efficacy ofthe result [113, 114]. As the technologyof scaffold design improves, the develop-ment may begin of synthetic pathwaysthat resemble tryptophan synthesis [102],polyketide synthesis [104] or carbamoylphosphate synthetase [105], where theenzymes are not merely co-located butactually channel the substrate down atunnel.

4.2Compartmentalization

Instead of recruiting enzymes to a scaf-fold and achieving a degree of control overthe diffusion of substrates from enzymeto enzyme of a pathway, it might beadvantageous to create a separate cellu-lar compartment to physically encapsu-late specific enzymes and substrates. An

impressive example of this has been seenin the investigations of Parsons et al. [5],who characterized the genes of a naturalbacterial organelle, which they called a bac-terial microcompartment (BMC). BMCsare polyhedral protein shells that are asso-ciated with specific biosynthetic pathways.The most well-studied BMC is the car-boxysome, which is associated with thefixation of carbon in Cyanobacteria [115].Parsons et al. focused their attention onthe pdu operon which contains a num-ber of genes, some of which are im-plicated in the construction of a BMC,while others are associated with Salmonellaenterica serovar. typhimurium LT2’s path-way for the conversion of 1,2-propanediolinto propionaldehyde, 1-propanol, andpropionyl-CoA. These enzymes, and thereactions they catalyze, are localized withinthe BMC that the pdu operon encodes[106]. Based initially on the sequence sim-ilarity to carboxysome proteins, and lateron the results of experiments where cer-tain proteins were subtracted, Parsons andcoworkers identified a set of five genes(PduA, PduB, PduJ, PduK, and PduN)which produced six proteins (PduB pro-duces two versions of the protein) that arenecessary and sufficient to produce emptyBMC compartments in E. coli, similar tothose produced by the native pdu operonin S. enterica serovar. typhimurium LT2. Al-though, not necessary, PduU was shownto help regulate the size of the BMC to theapproximately 100 nm dimensions it hasnatively. If the full set of required genesfor BMC construction was not present,then large intracellular structures suchas sheets, filaments, or hexagonal latticeswere observed. To prevent these structuresfrom forming, the wild-type order andorientation of the remaining Pdu genesneeded to be maintained. Lastly, by usingthe N-terminal region of a gene in the Pdu


operon, PduV , it was possible to create atag that localized GFP into the BMC.

Although Parsons et al. failed to demon-strate any enzymatic activity inside theresulting BMC, this was achieved in-side a compartment that was similar insome ways, but prepared from a cowpeachlorotic mottle virus capsid, albeit withthe very simple single enzyme system ofhorseradish peroxidase [116]. It was alsoshown possible to recruit compartmentsthat are already present in a cell, such asthe periplasmic space [117].

In keeping with the modular approachcentral to most Synthetic Biology methods,Parsons et al. and Deuber et al. devisedsystems by which tags could be fused toenzymes, allowing for their localizationand thus the spatial regulation.

5The Synthetic Cell

The organisms that have been engineeredthus far for industrial or other purposeshave had the advantage of being eas-ily manipulated genetically, because ofhigh transformation efficiencies and goodrecombination activities. Unfortunately,many industrially relevant organisms donot possess these characteristics and there-fore, cannot easily be engineered; for theseand other intractable organisms, novelengineering methods are necessary. Oneapproach, as adopted by the research teamat the J. Craig Venter Institute, has been tobuild a minimal cell that contains only es-sential genes, the functions of which havebeen characterized, in an attempt to un-derstand the basic principles of life. Sucha cell may also provide a base into whichvarious pathways can be placed to synthe-size industrially important products, butin a more energy efficient manner than

is possible with the presently engineeredorganisms.

During their efforts to build a minimalcell, the J. Craig Venter Institute group re-cently reported the creation of a bacterialcell that could be controlled by a chemi-cally synthesized genome [7]. The researchgroup described in detail the design, syn-thesis, and assembly of the 1.08 mega-basepair M. mycoides JCVI-syn1.0 genome (seeFig. 4), starting from digitized genomesequence information, and its transplan-tation into a recipient cell to create newM. mycoides cells that are controlled only bythe synthetic chromosome. To distinguishthe synthetic genome from the naturalgenome, the researchers placed ‘‘water-mark’’ sequences and included other de-signed gene deletions and polymorphismsin the synthetic genome (see Fig. 4). Eventhough the cytoplasm of the recipient cellis not synthetic, the research team referredto the cells produced after the transplanta-tion process as ‘‘synthetic cells,’’ becausethey are controlled solely by a genome thatwas assembled from chemically synthe-sized pieces of DNA. These synthetic cellshave expected phenotypic properties, al-though the JCVI-syn 1.0 transplants grewslightly faster than a control strain.

These studies, using several Mycoplasmaspecies, were the culmination of extensiveefforts over a number of years that ledto the development of several novel tech-nologies (as summarized in Fig. 5). First,as discussed above, the team developed astrategy of assembling viral-sized pieces toproduce large DNA molecules that allowedthem to assemble bacterial genomes inS. cerevisiae [40, 41, 43]. Second, the teamestablished additional methods to clonewhole bacterial genomes as centromericplasmids in yeast [118]. Third, they devel-oped methods to transplant the genomeof one bacterial species, M. mycoides ssp.


Overlapping DNA fragments(natural or synthetic) and a

yeast vector

Isolation

Genome witha yeast vector

TransformationTransplantation

Recipient cell

Resolution

Engineered bacteria

Insertion of yeast vectorinto bacterial genome

Methylation(if necessary)

Isolation

Genomeengineering

Fig. 5 Moving a bacterial genome into yeast,engineering the genome, and its re-installationinto a bacterium, by genome transplanta-tion. A yeast vector is inserted into a bacterialgenome by transformation, and the genome isthen cloned into yeast. Alternatively, a bacterialgenome is cloned by transforming overlappingDNA fragments (natural or synthetic) togetherwith a yeast vector into yeast and allowing thehost’s homologous recombination system toassemble an intact genome. After cloning, the

repertoire of yeast genetic methods is used tocreate insertions, deletions, rearrangements orany combination of modifications in the bacte-rial genome. This engineered genome is thenisolated and transplanted into a recipient cellto generate an engineered bacterium. Prior totransplantation, it may be necessary to methy-late the donor DNA in order to protect it fromthe recipient cell’s restriction system(s). Thiscycle can be repeated starting from the newlyengineered genome (dashed arrow).

capri, into a different recipient bacterial cellspecies, M. capricolum ssp. capricolum, toobtain cells of the donor M. mycoides ssp.capri [119]. These studies were extendedwhen the group was able to transplantthe genome of M. mycoides ssp. capri,which this time was isolated from yeastas a centromeric plasmid, into recipientM. capricolum ssp. capricolum cells and

produced viable M. mycoides ssp. capri cells[120]. The team also genetically altered theM. mycoides ssp. capri genome in yeast byusing the host’s powerful genetic tools andnewly developed tools [121] to produce anew strain of M. mycoides ssp. capri thatwould not have been possible with thetools currently available for these bacterialspecies.


Taken together, the Venter Instituteresearch team has developed a series oftechnologies that enabled them to clonewhole bacterial genomes, whether fromnatural sources or synthetic pieces, tomanipulate them, and to transplant themto produce viable bacterial cells (see Fig. 5).It will be interesting to see in the futurewhether this technology can be utilized forother intractable organisms.

6Societal Challenges Posed by SyntheticBiology

Along with the potential of significantbenefit, all new technologies raise societalconcerns. With respect to biotechnologyin general, these can be described asconcerns about bioterrorism, laboratorysafety, harm to the environment, thedistribution of benefits, and ethical andreligious concerns [122–125].

Synthetic Biology itself – both at the levelof research and in the application of suchresearch to the development of new prod-ucts – also raises a variety of societal con-cerns, some of which are identical to thoseraised by all biotechnology, though somemay be unique. These new or unique con-cerns may be especially important for thegovernance of the new technology. To itscredit, the community of research workersthat identify as synthetic biologists recog-nized at a very early stage that these societalconcerns were both real and legitimate.A good number of the synthetic biologistshave worked with a variety of policymakingand social science research communities,to ensure that the studies being carried outwould be performed in a safe and ethicalmanner. Given much interaction betweenand among these various communities,concerns that are unique – or that have

been recognized as of great importance,even if not unique – have been well ana-lyzed, and while the policy problems havein no way been solved the challenges havebeen very well articulated.

The first sets of societal concerns thatwere dealt with in detail by these com-munities were those of biosecurity andbiosafety. There is a constellation of ethi-cal issues (discussed below) that are of noless importance than security and safety.However, it was clear very early on thatif Synthetic Biology were to result in haz-ards that could not be mitigated for theresearch teams or for society as a whole,then there would need to be a morato-rium on such studies. Consequently, thesesocietal concerns regarding security andsafety were analyzed first by a varietyof policy researchers. Hence, the safetyand security analyses remain, for now,somewhat more advanced than the ethicsanalyses.

In particular as much of the recent Syn-thetic Biology studies have been conductedin a post ‘‘9–11’’ environment, concernsrelating to biosecurity were at the forefrontof virtually all policy analyses. Specifically,the ability to synthesize genomes meansthat, at least in some cases, access topathogens can no longer be physically lim-ited as long as the sequences are publiclyavailable. For now, the concerns are aboutincreasing the ease with which viruses,such as 1918 influenza, smallpox andEbola, can be obtained [125]. Additionally,Synthetic Biology may eventually provide arelatively straightforward way to constructpathogens with increased virulence, by al-lowing those with nefarious intent to adda variety of pathogenesis factors directly toa viral genome or bacterial chromosome.

In order to deal with these potential mali-cious applications, both the United StatesGovernment [126] and a consortium of


companies providing synthetic DNA [127]have released guidelines for the screeningboth of synthetic DNA orders, and of thecustomers placing these orders. Questionsas to whether these guidelines should belegally binding, the full range of what thecompanies should be screening for, andwhether orders for smaller pieces of DNA(oligonucleotides) should also be screenedare all currently under discussion.

Current biosafety or laboratory safetyconcerns are mostly focused on the speedand scale that Synthetic Biology bringsto research, and some concerns aboutworkers in the field who have not beentrained as microbiologists. At instituteswith a formal approach to dealing withbiosafety – such as universities, researchfoundations and scientific companies – in-stitutional biosafety committees and otherinstitutional groups will likely be taking upquestions in Synthetic Biology research inthe same way that they do all other bi-ological/biotechnological research. In anearlier report [125], policy researchers atthe JCVI (Michele Garfinkel and RobertFriedman), MIT (Drew Endy), and theCenter for Strategic and InternationalStudies (Gerald Epstein) have describedseveral options for such bodies for edu-cating themselves, and their institutionalresearch teams, about what steps must betaken to ensure that the research is safe.A more generic set of concerns about theresearch teams, and how to mitigate anypossible biosafety dangers, was discussedeven earlier in a report from the NationalAcademy of Sciences [128]. Yet another setof people interested in synthetic biologyhas also raised concern, namely the ‘‘do ityourself’’ (DIY) community [129]. Whilstfor the moment, little is being done by theDIY community that is clearly ‘‘syntheticbiology,’’ there has been much discussionamong the group eventually to employ

those technologies. In anticipation of thispossibility, the Presidential Commissionfor the Study of Bioethical Issues, whichis in the process of completing a reporton and recommendations for synthetic bi-ology, has addressed the issues of DIYspecifically [130].

Although the safety and security con-cerns may not be ‘‘solved,’’ it appears atleast that the great majority of issues havebeen laid out, and at least for the momentthere seem to be no risks that would lead tothe conclusion that the research should bebanned, or even severely restricted. (Thisalso appears to be the conclusion of thePresidential Bioethical Commission, al-though its current recommendations areonly in draft form.) Thus, the policy- andsocial science research communities haveturned at least some of their attention toother, broader societal challenges broughtabout by the potential use of SyntheticBiology technologies.

Concerns about harm to the environ-ment from accidental or planned releasesof engineered microbes date to discus-sions at the Asilomar meetings during themid-1970s. The two critical concerns arethat an engineered microbe will grow outof control if released accidentally or as partof a planned release, and that DNA from anengineered organism may be transferredto a related organism. These concerns havebeen dealt with over time via guidance andregulation dealing with the containment ofgenetically modified organisms and rulesfor testing these organisms for release intothe environment. Several US Governmentagencies are currently reviewing severalsets of regulations and guidance to un-derstand whether they are sufficient todeal with the use of many new microbesin open environments. For example, theNIH Guidelines for working with recom-binant DNA are currently being reviewed


to assure that guidance which was writtento deal specifically with recombinant DNAapplies equally to synthetic DNA [131].

In addition to the potential harm to peo-ple or to the environment (as discussedabove), there are societal challenges be-yond the physical. The distribution ofbenefits and risks is a very long-standingconcern, and one which surfaces for vir-tually every new technology. Ownershipas defined by intellectual property rights,the concentration of knowledge, and re-sources in a small number of firms orinstitutes – and how and whether theseresources should be shared – are is-sues that have become particularly acutearound research groups (both academicand commercial) who wish to developproducts. Interestingly, the Synthetic Biol-ogy community, in addition to civil societyorganizations, has placed this issue at theforefront of many of its own discussions[132]. These discussions could well leadto a better understanding of distributionconcerns and possible solutions generally.

Finally, hubris – sometimes called‘‘playing God’’ – might be the majornonphysical concern in Synthetic Biol-ogy, even if it is not fully unique in thiscase. In brief, concerns about hubris arefocused on a key issue: Are there ac-tions that human beings simply shouldnot take? In the case of constructing asynthetic cell, these questions arise formany communities, from religious tradi-tions to policymakers. Is constructing asynthetic cell creating life? If it is, is ithubris? If it is not creating life, then whatwould define creating life? In either case,is this hubris? And how might conduct-ing such experiments as constructing asynthetic cell change how human beingsthink of themselves, both individually andwith respect to other organisms and theenvironment in general? There are long

and thoughtful writings – both fiction andnonfiction, and both recent and deep inhistory – about hubristic pursuits that willnot be reviewed here. However, it shouldbe noted that these questions are beingstudied in detail with respect to SyntheticBiology by philosophers, ethicists and the-ologians [133].

7Concluding Remarks

Today, the capability is available to cre-ate any arbitrary DNA sequence, and toexpress that sequence in a wide varietyof living systems. By using modular partswith standardized structures, this DNAassembly capability has allowed the en-gineering of pathways, organelles, organ-isms, tissues, and even ecosystems. Unlikeprevious genetic engineering methods andselective breeding methods, these engi-neering projects have the potential to becheap, fast, and easy. As these capabilitiesare further refined and extended, an eracan be anticipated in which biological en-gineering will impact every industry andactivity of humankind.

Acknowledgments

The authors thank Dr. Carole Lartigue andDr. Chuck Merryman, and also the Syn-thetic Biology members of the J. CraigVenter Institute (JCVI) for their stimulat-ing discussions. Apologies are offered toany Synthetic Biology colleagues whosework has not been cited in this chapter.Funding for the research conducted at theJCVI was provided by Synthetic Genomics,Inc. and by the Office of Science (BER),U.S. Department of Energy, Grant No.DE-FC02-02ER63453.


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