GoldenBraid 2.0: A Comprehensive DNA Assembly · transfer of multigene structures to the plant...

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GoldenBraid 2.0: A Comprehensive DNA AssemblyFramework for Plant Synthetic Biology1[C][W][OA]

Alejandro Sarrion-Perdigones2, Marta Vazquez-Vilar2, Jorge Palací, Bas Castelijns, Javier Forment,Peio Ziarsolo, José Blanca, Antonio Granell, and Diego Orzaez*

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas (A.S.-P.,M.V.-V., J.P., B.C., J.F., A.G., D.O.), and Centro de Conservación y Mejora de la Agrodiversidad Valenciana(P.Z., J.B.), Universitat Politècnica de València, 46022 Valencia, Spain

ORCID ID: 0000-0002-4584-4827 (A.S.-P.).

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant syntheticbiology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigenestructures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describeGoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts forplant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modularcloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimizedcloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequentlyused modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interactiontools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database,tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication andmultigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements amongbioengineers to help implement plant synthetic biology projects.

Synthetic biology is producing a paradigm shift inbiotechnology based on the introduction of engineeringprinciples in the design of new organisms by geneticmodification (Check, 2005; Haseloff and Ajioka, 2009).Whereas synthetic biology has rapidly permeated mi-crobial biotechnology, the engineering of multicelledorganisms following synthetic biology principles isnow emerging and is mainly driven by the so-calledtop-down approaches, where newly engineered geneticcircuits are embedded into naturally existing organismsused as a “chassis.” The plant chassis offers an extra-ordinarily fertile ground for synthetic biology-like en-gineering. However, technology still faces the hugechallenge of performing engineering-driven geneticdesigns. One of the main technological challenges of

plant synthetic biology requires the construction andtransfer of multigene structures to the plant genome.This is putting pressure on developing DNA assemblyand transformation technologies adapted to plants.One main trend is the use of modular cloning, anengineering-inspired strategy consisting of the fabrica-tion of new devices by combining prefabricated standardmodules. In a modular strategy, predefined categories,the so-called “parts,” are assembled together followinga number of rules known as the “assembly standard.”Modular DNA building has been enthusiasticallyadopted by microbial synthetic biologists because it of-fers a number of advantages such as speed, versatility,laboratory autonomy, combinatorial potential, and oftenlower cost (Ellis et al., 2011). Modular methods acquirefull potential when parts are easily interchangeable andwhen one or a few assembly standards are shared bymany manufacturers.

A number of features define the value of a modularcloning method. Speed and efficiency are importantcharacteristics, as are also its simplicity and the abilityto produce scarless or scar-benign assemblies. More-over, any cloning strategy for synthetic biology shouldenable endless reusability; that is, it should ensure thatnew composite parts themselves can take part in newassemblies, therefore allowing unlimited growth. Sev-eral modular cloning strategies have been proposed inthe literature, and each presents advantages and short-comings. For instance, the original BioBrick standardwidely used in microbial synthetic biology scores amaximum for simplicity because a single rule governs

1 This work was supported by the Spanish Ministry of Economyand Competitiveness (grant no. BIO2010–15384), by a Research Per-sonnel in Training fellowship to A.S.-P., and by a Junta de Amplia-ción de Estudios fellowship to M.V.-V.

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Diego Orzaez ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

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all the assemblies (a property known as idempotency).However, it is not scar benign and is only relativelyefficient (Knight, 2003). Ligation-Independent Cloning(Aslanidis and de Jong, 1990), Uracil-Specific ExcisionReagent Fusion (Geu-Flores et al., 2007), and especiallyGibson Assembly (Gibson et al., 2009) are highly effi-cient DNA assembly methods, although they are nei-ther strictly modular nor widely adopted by plantbiotechnologists. In sharp contrast, Gateway Cloning(Hartley et al., 2000) is of widespread use in plantlaboratories (Karimi et al., 2002, 2007; Estornell et al.,2009). Recently, MultiRound Gateway technologiesopened Gateway capabilities to the sequential deliveryof multiple transgenes by multiple rounds of recom-bination reactions (Chen et al., 2006; Vemanna et al.,2013). In general, Gateway-based technologies arehighly efficient. Unfortunately, they are not alwaysscar benign, as they leave 21-bp scars between build-ing blocks. Other technologies involving rare cutters orhoming endonuclease-based strategies have also beendeveloped and adapted to plant transformation (Linet al., 2003; Dafny-Yelin and Tzfira, 2007; Fujisawaet al., 2009), including combinations of homing endo-nucleases and engineered zinc finger nucleases (Zeeviet al., 2012) and iterative in vivo assembly rounds ofCre recombinase and phage1 site-specific recombina-tion (Chen et al., 2010). Many of these techniques canserve as efficient assembly methods for multigene engi-neering. Nonetheless, a prerequisite to become a stan-dard for plant synthetic biology is the development of aset of rules and tools based on those technologies thatcan be shared by as many laboratories as possible.Recently, a very powerful DNA assembly method

named Golden Gate was described (Engler et al., 2008,2009). Golden Gate uses type IIS restriction enzymes togenerate four-nucleotide sticky ends flanking eachDNA piece, which can be subsequently joined togetherefficiently by T4 ligase. The assembly reaction is mul-tipartite and is performed in a single-tube reaction toyield highly efficient scarless or scar-benign assem-blies. This is because type IIS recognition sites areeliminated upon ligation, leaving only four-nucleotideseams, which can be user defined. These features makethe Golden Gate technology an excellent candidate toset up a standardized modular cloning system. How-ever, as originally conceived, Golden Gate is not areusable system and, therefore, cannot be used effi-ciently for multigene engineering.Most recently, two strategies were described to en-

able the reusability of the Golden Gate cloning scheme:MoClo (Weber et al., 2011a) and GoldenBraid (Sarrion-Perdigones et al., 2011). Both methods use the multi-partite Golden Gate property to build transcriptionalunits (TUs) starting from basic standard buildingblocks, and both create specially designed destinationvectors to enable Golden Gate-built TUs to be assem-bled among them. Whereas the GoldenBraid mini-malist cloning strategy allows multigene growth byenabling binary assemblies between TUs, the MoClodestination vectors offer the interesting possibility of

performing multipartite assembly at the TU level, al-beit at the cost of the higher complexity of its vectortoolkit.

The Golden Gate-based strategies MoClo andGoldenBraid are ideal to serve as modular assemblysystems in plant synthetic biology, as they are efficient,reusable, and scar benign. To realize their full poten-tial, it is very important to (1) advance in adoptingcommon standards, so building blocks can be sharedby as many users as possible; (2) further optimize thedesign of cloning strategies to improve speed and ef-ficiency; and (3) improve the user experience by gen-erating new hardware (building blocks and modules)and software (databases and assembly programs) toolsthat simplify and facilitate the engineering process.

To facilitate the implementation of plant synthetic bi-ology approaches, we present GoldenBraid 2.0 (GB2.0), anew version of the GoldenBraid cloning strategy. In thisnew version, we defined, in concert with MoClo de-velopers, a common assembly standard by establishingarbitrary, yet scar-benign, assembly seams within a TUthat facilitate part exchangeability. In addition, weoptimized the versatility of the GoldenBraid strategy byenhancing its minimalist design, creating a universal partentry vector and simplifying the cloning setup. Finally,we generated a collection of premade genetic modulesand new software tools for the purpose of facilitating thebuilding of frequently used genetic structures. Inshort, we present a new grammar for plant syntheticbiology and introduce a comprehensive toolkit to fa-cilitate the use of GB2.0 in composing new geneticdesigns.

RESULTS

The GB2.0 Cloning Strategy

To describe the GB2.0 assembly strategy, we follow ananalogy with a natural language because we believe thiscomparison closely describes the GB2.0 cloning strategystructure and facilitates its understanding. This isbecause the hierarchical manner in which the differ-ent building blocks in GB2.0 are combined to form amultigenic structure become analogous to the waygrammar elements (morphemes, words, phrases, andsentences) are combined hierarchically to create acomposition. Figure 1A provides an equivalence tablebetween the elements of English grammar and theelements of the GB2.0 system.

GBparts: Words and Phrases. Definition of the GB2.0 Grammar

The first task in upgrading GoldenBraid was to de-fine the minimal standard building blocks in GB2.0,the so-called GBparts, which can be considered the“words” of the GoldenBraid grammar. GBparts arefragments of DNA flanked by four-nucleotide over-hangs. They are stored as inserts within a speciallydesigned entry vector (pUPD), from where they are re-leased by cleavage with BsaI or BtgZI restriction enzymes

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to generate the corresponding flanking overhangs.GBparts are classified into different classes or cate-gories according to their specific functions. EachGoldenBraid class is defined by its flanking four nu-cleotides, which will overhang upon enzyme digestionand will determine its position within the TU. Wedefined 11 standard classes (Fig. 2A) that correspondto the basic functional categories in a typical TU. Thefirst three categories (01, 02, and 03) were orderly set inthe 59 nontranscribed region and correspond to oper-ators and promoter regions. Next, we defined sevencategories in the transcribed region: one correspondedto the 59 untranslated region (UTR; 11); one related tothe 39 UTR (17); four were reserved to the coding re-gion (13–16); and an additional class was set as a bufferzone to facilitate, among other designs, the construc-tion of noncoding TUs intended for gene silencing.Lastly, we set a final class (21) for standard 39 untran-scribed GBparts.

Besides the basic classes, GB2.0 also employs “super-classes.” For practical purposes, it is convenient to groupseveral contiguous basic GBparts that, together, perform

a defined function (e.g. a complete promoter or a fullcoding region) in a single DNA element (a Golden-Braid superpart, abbreviated to GBSpart) instead ofsplitting it into its basic standard parts. This isanalogous to an English phrase, which comprises agroup of words that function as a single unit withinthe hierarchical structure of the sentence syntax (e.g.a subject or a direct complement). As with GBparts,GBSparts are ultimately DNA fragments storedwithin the pUPD vector. Upon digestion with BsaI orBtgZI, the whole phrase is released as a solid indi-visible unit flanked by four-nucleotide “barcodes.” Inpractice, GBSparts are very convenient, as they re-duce the number of elements that need to be assem-bled to produce a TU; therefore, they enhanceefficiency. Frequently used superclasses are depictedin Figure 2, B and C. For example, the promoter re-gions normally employed in traditional cloning cor-respond to the superclass (01–12). GBparts andGBSparts are components of the GoldenBraid col-lection, and their sequence information is stored inthe GBdatabase.

Figure 1. Analogies between GB2.0 and English grammar. A, GB2.0 elements can be compared with those of a natural lan-guage. In English grammar (left), morphemes are joined together to make words; words are combined together to make phrasesand sentences, which are further joined to make a composition. In GB2.0 (right), the simplest units are GBpatches, used to buildany of the 11 standard GBparts. GBpatches can be also combined in GBSparts to facilitate cloning (e.g. a whole promoter).GBparts and GBSparts are combined in a multipartite reaction to build TUs, which can be used for plant transformation, or canbe reused and combined with other TUs to build multigene modules. B, Flow chart of the GoldenBraid assembly steps. It startswith the GoldenBraid domestication of GBpatches into GBparts or GBSparts; GBparts are multipartitely combined to build upTUs; finally, TUs are binarily assembled to build modules and multigene constructs. [See online article for color version of thisfigure.]

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GBpart Domestication: Creating Words and Phrases

The process of adapting a DNA building block (GBpartor GBSpart) to the GoldenBraid grammar is referred toas domestication. GoldenBraid domestication usuallyinvolves PCR amplification of the target DNA (wordor phrase) using GB-adapted primers (for details, seeFig. 3) and the subsequent cloning of the resultingPCR fragment into the pUPD vector using a BsmBIrestriction-ligation reaction. Occasionally, domestica-tion may involve the removal of internal BsaI, BsmBI,or BtgZI restriction sites. In order to facilitate aneventual automation of the cloning process, the GB2.0system includes a standard procedure for internalsite removal. This procedure, described in detail inSupplemental Figure S1, involves the amplification of thetarget DNA in separated fragments (named GBpatches)using GB-adapted primers, which incorporate single

mismatches to disrupt the enzyme target sites. Onceamplified, GBpatches are reassembled together in a single-tube BsmBI restriction-ligation reaction into pUPD to yielda domesticated GBpart or GBSpart.

The GB2.0 Destination Plasmids Kit

GoldenBraid destination vectors (pDGBs) are binaryvectors that function as recipients of new assemblies.Each pDGB contains a GBcassette (the selection LacZgene flanked by two restriction/recognition sites cor-responding to two different type IIS enzymes; Fig. 4A).In addition, GB2.0 plasmids include a watermark (i.e. adistinctive restriction site flanking the GBcassette) to helpplasmid identification. Detailed information about thesequences of the different GBcassettes is also provided inFigure 4A. The special orientation and arrangement ofthe restriction enzymes define two levels of pDGBs, the

Figure 2. The complete GB2.0 grammar and its most frequently used structures. A, Schematic overview of a TU structure wherethe 11 standard GoldenBraid classes are depicted: 01, 02, and 03 GBparts form the 59 nontranscribed region (59NT); position 11is the 59 UTR of mRNA; 12 is a linker region; 13 to 16 (TL1–TL4) are four divisions of the translated region; 17 is the 39 UTR ofmRNA; and 21 is the 39 nontranscribed region of the TU (39NT). B, Frequently used structures for the protein-coding TUs. Theelements forming each frequently used structure and the class that they belong to are depicted. C, Frequently used structures forRNA silencing, including amiRNA, hpRNA, and tasiRNA. 59NTand 39NTas well as 59UTR and 39UTR are defined above; LINKrepresents a region between the 59 UTR and the coding sequence where tags or fused proteins can be placed; PROM is apromoter; CDS is the coding DNA sequence; TER represents the terminator; SP is signal peptide; NT and CT are N- andC-terminal tags or fusion proteins; OP is a promoter operator; minPROM is a minimal promoter; 59FS and 39FS indicate theflanking sequences of the amiRNA precursor sequences; Target represents the region of the amiRNA structure comprising theloop and the complementary target sequences; GOI and IOG are the fragments of the gene of interest in an inverted orientation;INT is the intron for hpRNA processing; mir173 represents the mir173 target site for tasiRNA processing; fGOI indicates thefragment gene of interest to be silenced. [See online article for color version of this figure.]


GoldenBraid 2.0


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a-level and V-level plasmids, which are used for theBsaI- and BsmBI-GoldenBraid reactions, respectively.Plasmids also differ in the resistance marker that is as-sociated with each level (kanamycin for level a andspectinomycin for level V, allowing counterselection).To ensure an endless cloning design, a minimum set offour pDGBs is required (pDGBV1, pDGBV2, pDGBa1,and pDGBa2). Additionally, this set can be expanded toeight plasmids to enable assemblies in different orienta-tions (pDGBV1R, pDGBV2R, pDGBa1R, and pDGBa2R).For GB2.0, we constructed two complete sets of pDGBs,one based on the pGreenII backbone and another setbased on the pCAMBIA backbone. The sequence in-formation of all 16 pDGBs in GB2.0 is uploaded in theGBdatabase.

The Composing Strategy: from Single Words toFull Compositions

The GB2.0 cloning strategy comprises two types ofassemblies (see the GB2.0 chart in Fig. 1B): multipartiteassemblies and binary assemblies. Multipartite assem-blies are performed to create single TUs. The differentGBparts and GBSparts required to produce a well-constructed TU are mixed together in a single tube inthe presence of a pDGB, the corresponding type IISrestriction enzyme(s), and the T4 ligase, and they are

incubated in cyclic restriction-ligation reactions. If allthe elements are correctly set in the reaction, they or-derly assemble within the destination vector and gen-erate a so-called expression vector, which harbors theassembled composite part. Our pDGBs are binary vec-tors; therefore, the resulting expression clone is ready tobe used directly for Agrobacterium tumefaciens-mediatedplant transformation.

After building a new TU using a multipartite as-sembly, the resulting new expression clone can bebinarily combined with another expression clone toproduce increasingly complex multigene structuresanalogous to how sentences are combined to createa written composition. The solution provided byGoldenBraid cloning relies on the special design ofGoldenBraid destination vectors, which introduces adouble loop (braid) into the cloning strategy. A compositepart (a TU or a group of TUs) cloned in a given entryvector can be combined only with a second composite partcloned in the complementary entry vectors at the samelevel. This is done in the presence of a destination vectorof the opposite level and generates a new expressionvector at the opposite level. A formal notation de-scribing the rules for multipartite and binary assem-blies is shown in Figure 4, B and C.

By choosing appropriate combinations of expres-sion and destination vectors, it is possible to create

Figure 3. Standardized domestication ofGBparts. GBparts are domesticated by ampli-fying the desired sequence with standardGBprimers (GB.F and GB.R). GBprimers includeapproximately 20 nucleotides of the gene-specific primer (GSP) and a tail region that in-cludes a BsmBI recognition site, the cleavage sitefor cloning into pUPD, and the four-nucleotidebarcode (1234 and 5678). The amplified DNApart is cloned into pUPD in a restriction-ligationreaction, with BsmBI as the restriction enzyme.The resulting GBpart is cleavable by BsaI andBtgZI to produce 1234 and 5678 flanking over-hangs. BsmBI recognition sequences are depictedin orange in the DNA sequence; BsaI and BtgZIare labeled in red and blue, respectively. Enzymecleavage sites are boxed. [See online article forcolor version of this figure.]





increasingly complex structures, and the only limitsare the capacity of the vector backbone or the biolog-ical restrictions imposed by bacteria. Moreover, all thenew composite parts are fully reusable (they can beused directly for part transformation or can beemployed in new assemblies) and exchangeable (theycan be combined with the GoldenBraid modules that

are produced separately in different laboratories byfollowing the same assembly rules).

Innovative Features in the GB2.0 Cloning Strategy

Besides a proposal for a grammar, GB2.0 introducesa number of new elements that modify the original

Figure 4. GB2.0 cassettes and assembly rules. A, GB2.0 cassettes and their comparison with the previous GoldenBraid version.GBcassettes comprise a LacZ selection cassette flanked by four type IIS restriction sites (BsaI and BsmBI) positioned in inverseorientation. The previous GoldenBraid plasmid kit comprised four destination plasmids, two in each assembly level. GB2.0incorporates four additional plasmids that permit the assembly of transcriptional units in reverse orientation using the sameGBparts. Additionally, the six four-nucleotide barcodes of GoldenBraid (A, B, C, 1, 2, and 3) collapsed in only three GB2.0barcodes, where A ≡ 1, B ≡ 2, and C ≡ 3. This special design feature permits GBparts to be directly assembled in both levelplasmids. Finally, GB2.0 plasmids incorporate distinctive restriction sites flanking the GBcassette as watermarks for plasmididentification. BsaI cleavage sequences are boxed in red, BsmBI cleavage sequences are boxed in orange, and sites where bothenzymes can digest are boxed in green. The watermark restriction sites are underlined. B, Rules for multipartite assembly. ThepUPD elements represent each GBpart and GBSpart that conforms to a grammatically correct TU, pDGBVi is any level Vdestination vector, pDGBai is any level a destination vector, and pEGBVi (X) and pEGBai (X) are the resulting expressionplasmids harboring a well-constructed transcriptional unit X. C, Rules for binary assembly. (Xi) and (Xj) are composite partsassembled using the multipartite assembly option; (Xi+Xj) is a composite part of (Xi) and (Xj) that follows the same assemblyrules as (Xi) and (Xj); pEGBa1(X), pEGBa2(X), pEGBV1(X), and pEGBV2(X) are expression plasmids hosting a composite part X;and pDGBV1, pDGBV2, pDGBa1, and pDGBa2 are destination plasmids hosting a LacZ cassette. [See online article for colorversion of this figure.]


GoldenBraid 2.0



GoldenBraid cloning design to make it simpler andmore versatile. Many of the new GB2.0 features rely onthe design of the plasmid that harbors GBparts andGBSparts, the Universal Domesticator (pUPD). ThepUPD cassette is designed to serve as a polyvalententry vector for all the different GBparts and GBSparts,regardless of their category. This is because the four-nucleotide barcodes are incorporated into the GBpartby PCR instead of being imprinted in the plasmiditself. Such a universal plasmid enables us to establish asingle standard protocol for all the domestication partsbased solely on its sequence information and categoryspecification.

Another innovative feature of pUPD is the incor-poration of both BtgZI and BsaI sites flanking theGoldenBraid cassette. The enzyme target sites arearranged in such a way that both BsaI and BtgZI di-gestions release exactly the same piece of DNA, whichcontains the same four-nucleotide overhangs, regard-less of the enzyme used. This opens up the possibilityof GBparts being assembled into the a- and V-levelvectors indistinctly by using either BsaI reactions andBtgZI/BsmBI reactions, respectively. To enable thisoption, the GoldenBraid cassettes in the pDGBs havealso been redesigned and simplified. In the previousversion, the sequences of the restriction sites for BsaI(named A, B, and C) differed from the restriction sitesfor BsmBI (named 1, 2, and 3). In GB2.0, we made A ≡1, B ≡ 2, and C ≡ 3 (for details, see Fig. 4A). In thisway, and by making full use of the dual BsaI/BtgZIrelease from pUPD, any pDGB can be used as a re-cipient of a multipartite assembly, which, therefore,makes entry in the GoldenBraid loop fully symmetric.Thus, BsaI reactions are performed to build TUs ina-vectors, and BsmBI/BtgZI reactions (BsmBI to openpDGB and BtgZI to release the GBpart) are performedto build TUs in V-pDGBs. Furthermore, by choosingany of the reverse pDGB plasmids as recipients, TUorientation can be inverted. This opens up the possi-bility of creating new binary assemblies in all the possiblerelative orientations.

The pUPD design provides yet another interestingnew feature to GB2.0, as it enables the use of a non-standard assembly level operating below the standardGBpart level (referred to as the GBpatch level). Thisfeature can be most convenient for a number of ap-plications, including the generation of seamless junc-tures, introducing combinatorial arrangements into proteinengineering, or promoter tinkering using nonstandardpositions. The process is similar to the above-describeddomestication procedure. An example of the use of theGBpatch level for combinatorial antibody engineeringis depicted in Supplemental Figure S2.

Frequently Used Structures

There are a limited number of structural types forthe majority of synthetic transcriptional units and ge-netic modules. For instance, many protein-encodingTUs can be constitutively expressed, whereas others

are regulated by 59 (or 39) operators. The resultingproteins can be preceded by a signal peptide or maycontain C-terminal and N-terminal fusions. Besides,noncoding TUs can be used for silencing purposes. Tocope with this functional diversity while simplifying theuser toolbox, we defined a group of “frequently usedstructures,” for which specific prearranged GBparts andGBSparts were developed (Fig. 2, B and C). We now goon to describe some of the frequently used structuresthat are currently included in the GoldenBraid systemand their associated tools.

Basic Expression Cassettes for Multigene Engineering

Multigene engineering may require the use of differ-ent regulatory regions to avoid the silencing associatedwith the repeated use of a DNA sequence in the sameconstruct. To meet this requirement, we incorporatedseveral regulatory 59 and 39 regions into the GB2.0 col-lection. Most 59 regulatory regions are (01–12) GBSpartscomprising a promoter and 59 UTR, whereas 39 regula-tory regions are (17–21) GBSparts comprising 39 UTRand terminator regions. According to this basic setup,full (13–16) open reading frames can be easily incorpo-rated into tripartite reactions to obtain constitutivelyexpressed TUs. In order to undertake synthetic biologyprojects, it is very important to have a range of regula-tory regions available and that the expression strengthprovided by each promoter/terminator combination isproperly characterized so that the multigene expressioncan be adjusted accordingly. As a first approach towardthe characterization of a set of basic expression cassettes,we finely characterized the relative promoter/terminatorstrength of a number of cassettes using the Renilla/luciferase system (ratio determined by the Dual Glo As-say System [Promega]) in transiently transformed Nico-tiana benthamiana leaves. The characterization of (01–12)and (17–21) regions as individual entities is a rela-tively straightforward procedure using GB2.0 cloning.However, as the collection grows, the individual char-acterization of all the possible combinations becomesan intractable task. Therefore, we decided to investigateto what extend the transcriptional strength provided byeach “promoter/terminator” (i.e. 01–12_17–21) combi-nation can be inferred from the separated contributionof each region. For this purpose, all the (01–12) pro-moter regions in the collection were tested by theRenilla/luciferase system in combination with a com-mon (17–21) terminator region (TNos). In parallel, allthe (17–21) terminator regions in the collection weretested in combination with a common (01–12) pro-moter region (PNos). The “experimental transcriptionalactivity” (ETA) of each region was calculated as beingrelative to the Renilla/luciferase values of a (01–12_17–21)reference combination (PNos_TNos), which was arbi-trarily set as 1 (for construct details, see Fig. 5A). The ETA(01–12) values ranged between 0.47 6 0.01 and 15.03 61.44 relative luminescence units, whereas the ETA (17–21)values ranged between 0.776 0.18 and 2.616 0.54 (Fig. 5,B and C). Using these data, “theoretical transcriptional






activity” (TTA) was calculated for each cassette combi-nation (Fig. 5D) as the product of the individual ETAs ofthe two regulatory regions. Finally, the Renilla/luciferaseratio of a number of cassette combinations (covering 65%of total possibilities) was also tested experimentally. Aswe can see in Figure 5E and Supplemental Figure S3,there is good agreement between the theoretical and ex-perimental activity values. Of the 34 experimental com-binations assayed in the evaluation test, 31 showeddeviation in relation to the theoretical values below2-fold (60.3 in logarithmic values; for detailed infor-mation, see Supplemental Fig. S3C).

Regulated Expression Cassettes

The GoldenBraid grammar contains several standardpositions for the insertion of regulatory regions. In the 59untranscribed region, we defined three standard GBpartsto allow combinatorial promoter tinkering and to fa-cilitate the insertion of synthetic operators. As a func-tionality proof, we assembled and tested the premadecassettes for heat shock and dexamethasone-regulatedexpression; the latter is based on the “operated promoterA” scheme shown in Figure 2B. The Renilla/luciferase/p19 reporter cassettes constructed with promoters pHSP70and pHSP18.2 showed clear induction after incubationat 37°C (Supplemental Fig. S4). The potential of theGoldenBraid modular assembly was further demonstratedwith the construction of two regulated systems basedon the fusion of the glucocorticoid receptor with theDNA-binding domains of LacI or Gal4 and the acti-vation domain of Gal4. In this transactivation exam-ple, up to 15 premade modules comprising codingand noncoding regulatory regions were efficiently as-sembled de novo to produce two operated luciferaseTUs, which clearly responded to the presence of dexa-methasone (Supplemental Fig. S5).

Protein-Protein Interaction Tools

Reporter fusion partners are powerful analytical toolsutilized in the study of protein-protein interactions.However, the use of unlinked cotransformation for thedelivery of the interaction partners often compromisesthe extraction of reliable qualitative data, based on thepoorly supported assumption that cotransformationefficiency in each cell is the same for all fusion part-ners. We reasoned that the linked cotransformation offusion partners can help improve the sensitivity andaccuracy of the protein-protein interaction analysis.Bearing this use in mind, we designed premade modulesfor the bifluorescence complementation (BiFC) assays.For this purpose, BiFC adaptors with a (01–12) structurewere constructed containing the full 35S promoter andthe corresponding yellow fluorescent protein fusionpartners. Based on this setup, bait and prey with acanonical (13–16) structure can be easily assembled inmultipartite reactions to form the required fusionproteins. The prearranged BiFC tools were functionallytested using transcription factors Akin10/Akinb2 as

positive interaction partners and a spermidine syn-thase as a negative partner (Belda-Palazón et al., 2012).As observed in Supplemental Figure S6, the number ofcells showing positive interactions with the GB-assistedlinked cotransformation setup outnumbers those of theunlinked cotransformation approach.

Silencing Tools

The negative regulation of endogenous genes oftenproves an engineering requirement. For this reason,special frequently used structures were defined for threeRNA-silencing strategies: transacting small interferingRNA (tasiRNA), artificial microRNA (amiRNA), andhairpin RNA (hpRNA; Supplemental Fig. S7A). Detailsof all the elements used in the RNA interference designsare provided in Supplemental Table S1.

For the generation of tasiRNA constructs, special (01–11) GBSparts containing the mir173 trigger sequence arerequired. A cauliflower mosaic virus (CaMV) 35S-basedGBSpart for constitutive tasiRNA expression is currentlyavailable in the GoldenBraid collection. Regulated ortissue-specific tasiRNA expression can be designedusing the GBpatch special feature of GB2.0. For thefunctional characterization of the tasiRNA structure, a410-bp fragment of Arabidopsis (Arabidopsis thaliana)phyotoene desaturase (PDS; Felippes et al., 2012) wasincorporated as a (12–16) GBSpart and was trans-formed into Arabidopsis to yield approximately 0.1%seedlings with the albino phenotype (SupplementalFig. S7C). The tasiRNA constructs require the coex-pression of miR173 for effective silencing in plantspecies other than Arabidopsis (Felippes et al., 2012).To extend the species range of the tasiRNA tool, a newTU with a constitutively expressed miR173 was con-structed and incorporated into the collection. Thefunctionality of the dual construct was tested tran-siently in N. benthamiana using PDS as the silencingtarget, which resulted in the bleaching of the infil-trated area (Supplemental Fig. S7D).

An amiRNA silencing tool was also enabled withthe creation of two special GBSparts, namely 59FS and39FS. These GBSparts require noncanonical barcodes toallow the seamless assembly of 59FS and 39FS in theamiRNA precursor. The special categories are denotedas (12–13B) and (16B), respectively, where B indicatesthe four noncanonical flanking nucleotides (GTGA andTCTC, respectively). The standard (01–11) promoterswithout ATG and the (17–21) terminators were used inthe amiRNA design. The central region (14B–15B),containing a fragment of the gene target sequence, wasconstructed using gene-specific oligonucleotides, asdescribed in Supplemental Figure S7B. In order tovalidate the proposed structure, Arabidopsis PDS silencingwas assayed using a gene target fragment that was for-merly described by Yan et al. (2011; Supplemental Fig. S7E).The resulting amiRNA construct was transformed intoArabidopsis, yielding seedlings with the albino phenotype.

Finally, in the hpRNA structure, the regulatory re-gions lacking ATG are inserted as (01–11) parts. An


GoldenBraid 2.0















Figure 5. Characterization of regulatory regions for basic expression cassettes. A, Constructs for ETA quantification. The pro-moter (01–12)i_(17–21)TNos constructs comprise a first TU with the (01–12) promoter of interest, the firefly luciferase and thenopaline synthase terminator, followed by the Renilla reference module (top row). For the (01–12) TNos _(17–21)j terminatorconstructs, the first TU comprises the (17–21) terminator of interest and the firefly luciferase and the nopaline synthase promoter(middle row). For activity normalization, the PNos:luciferase:TNos construct combined with the Renilla reference module wasused (bottom row). B, The ETA of the promoter regions in (01–12)i_(17–21)TNos constructs was determined as the firefly (FLuc)/Renilla (RLuc) luciferase activity ratio of each construct normalized with the equivalent ratio of the PNos:luciferase:TNosconstruct. Error bars represent the SD of at least three replicates. C, The ETA of terminator regions in the (01–12)PNos_(17–21)jconstructs was estimated as described in B. D, Scheme of the combinatorial promoter/terminator constructs comprising a firstTU with a (01–12) promoter, the firefly luciferase, and a (17–21) terminator and combined with the Renilla reference module. E,Correspondence between the ETA and TTA data in the combinatorial constructs. The logarithm of the ratios between the ETAand TTA values for 34 experimental promoter/terminator combinations is plotted. Bars represent average values 6 SD. PNos,nopaline synthase promoter; TNos, nopaline synthase terminator; P35s, CaMV 35S promoter; p19, Tomato Bushy Stunt Virussilencing suppressor. [See online article for color version of this figure.]





intron from tomato (Solanum lycopersicum; SGN-U324070) was incorporated into the collection to serveas a (14–15) intron GBpart. The inverted fragments ofthe target gene of interest can be cloned at positions(12–13) and at position (16).

GW-GB Adapter Tool

A GW-GB adapter tool was incorporated into theGB2.0 collection in order to facilitate the transitionbetween the Gateway and GB2.0 assembly methods(Supplemental Fig. S8). GW-GB adapters are GBpartsor GBSparts [e.g. a (12–16) GBSpart to adapt codingregions] made of a Gateway cassette flanked by attR1-attR2 sites and embedded inside the pUPD plasmid.As such, adapter vectors can be used directly as des-tination plasmids for Gateway entry clones flanked byattL1-attL2 sites. In this way, Gateway entry clones canbe transferred individually or in bulk to the pUPDplasmid and become ready-to-use GBSparts. Alterna-tively, the GW-GB adapter can be employed as anordinary GBSpart to create a new multigene constructin a binary vector. Consequently, the resulting multi-gene construct becomes a Gateway destination vectorcontaining an attR1-attR2 Gateway cassette, whereGateway entry clones can be inserted individually orin bulk. It should be noted that direct Gateway-to-GB2.0 adaptation does not remove internal enzymetarget sites; therefore, the efficiency of subsequent as-sembly reactions can lower.

GoldenBraid Collection and Software Tools

When this paper was being written, our in-houseGoldenBraid collection contained more than 400 en-tries. As the collection grows, engineering is becomingincreasingly easy and fast, because, on occasion, therequired GBparts, GBSparts, and TUs are already do-mesticated and/or constructed. To efficiently handlethis collection, we developed a Web framework thathosts a GBdatabase and offers software tools to facil-itate the assembly process. The GB2.0 Web site wasimplemented using Django, a Python Web frameworkthat supports rapid design and the development ofWeb-based applications (version 1.5; Django SoftwareFoundation; http://djangoproject.com). The object-relational database management system PostgreSQLwas chosen to host our schema, which allowed incor-poration of the sequences of all the elements includedin the collection. Additional relevant information on partidentity, functionality, and indexing is also provided.Given the simplicity of the GoldenBraid assembly

rules, it was relatively straightforward to develop soft-ware tools that assist in GB2.0 assembly. Therefore, wedeveloped a software package comprising three pro-grams, each program corresponding to one of the threebasic processes in GB2.0 assembly. The first program,named GBDomesticator, assists the part adaptationprocess to the GoldenBraid standard. It takes an inputDNA sequence provided by the user and offers the best

PCR strategy to remove internal enzyme target sitesand to add flanking nucleotides to it according tothe specified category. A second program, known as theTUassembler, takes GBparts and GBSparts from thedatabase and simulates a multipartite assembly in sil-ico. The TUassembler includes shortcuts to frequentlyused structures assembly as well as a free-hand option.Finally, a third program, BinaryAssembler, performsin silico binary assemblies between the composite partsstored in the GoldenBraid database. BinaryAssembleroffers the possibility of choosing the relative orientationof each member of the assembly. All three programsgenerate a detailed laboratory protocol to perform thedomestication/assembly and to return a GenBank-formatted file containing the final domesticated/assem-bled sequence. The GoldenBraid database and softwaretools are available at http:/www.gbcloning.org.

DISCUSSION

The aim of this work is to provide a standard frame-work for DNA assembly in plant synthetic biology. We,and others, realized that the modularity of the mul-tipartite assembly based on type IIS enzymes offers agreat opportunity for standardization by following apositional information scheme that resembles the gram-mar of a sentence in many natural languages. Indeed, it isillustrative to conceive the transcriptional unit as a similarstructure to a sentence, which is made up of hierarchicallyassembled elements like morphemes, words, and phra-ses. It is also interesting to envision the whole engineeringprocess as a way to imprint instructions using DNAstrings. Therefore, we, in concert with MoClo developers,propose a common grammar where the four-nucleotideoverhangs are predefined for each position within thetranscriptional unit. Overhang assignation is mainly ar-bitrary, but some decisions are made to make them scarbenign. For instance, the 12-13 boundary defining thebeginning of the coding DNA sequence was designed toinclude the start codon, whereas the 13-14 boundary wasmade compatible with signal-peptide cleavage sites.

In our view, this new GB2.0 cloning scheme has anumber of features that make it a good candidate fora plant assembly standard. Many of those features areconsubstantial to the Golden Gate system: very highefficiency, modularity, and the ability to produce scare-benign assemblies. GB2.0 also incorporates the reus-ability and modularity of the GoldenBraid and MoClosystems and goes beyond them in that it provides astandardized framework, goes deep into the versatilityand the minimalist design of the GoldenBraid loop, andincorporates new tools to assist cloning.

A major drawback of defining a standard is loss inversatility, since no standard can cope with all customdesign requirements. To deal with this problem, weincorporated an underlying nonstandard assemblylevel that makes full use of the newly designed pUPDvector. At this level, nonstandard GBpatches can becustom designed, for instance, for scarless assembly by


GoldenBraid 2.0



http://djangoproject.com

http://www.gbcloning.org


choosing the appropriate four-nucleotide overhangs.GBpatches are assembled together into standard GBpartsor GBSparts. We made full use of the GBpatch level forBiFC, amiRNA, and antibody engineering. Other pos-sible uses include promoter tinkering or nonstandardcombinatorial assemblies within the coding DNA se-quence, as exemplified in the construction of custom-ized Transcription Activator-Like effectors (Weber et al.,2011b; Li et al., 2012). Additionally, the GBpatch level isused for GBpart domestication (i.e. for the removal ofinternal enzyme recognition sites). This feature is alsoenabled by the special design of the new entry vectorpUPD, which introduces inversely oriented BsmBI sitesinto the GoldenBraid cassette. This new design turnsthe pUPD plasmid into a universal entry vector, as thefour nucleotides conferring part identity are not locatedin the entry vector, as they are in previous designs(Weber et al., 2011a). Instead, in the present setup, the four-nucleotide barcode is incorporated into the primers usedduring initial part/patch isolation. As a toll, this strategyinvolves the requirement of longer PCR primers duringinitial part isolation. This minor drawback is by far com-pensated by the simplicity introduced by the universaldomesticator: in the absence of this solution, a mini-mum of 11 different entry vectors would be requiredto harbor the different categories in the GoldenBraidgrammar, along with an unaffordable amount of addi-tional vectors to allow the formation of all the possible“phrasal” combinations.

The underlying GoldenBraid cloning pipeline hasbeen substantially simplified in the GB2.0 version to

reduce redundancy and to achieve a minimalist design.Figure 6 depicts the comparison of GB2.0 and the pre-vious GoldenBraid structure. Once again, most of theimprovement achieved stems from the specific designof the new entry vector pUPD. First, the asymmetry ofthe cloning loop is corrected in GB2.0 with the intro-duction of a BtgZI site into the entry vector. BtgZI is aspecial enzyme that cuts 10 nucleotides away from itsrecognition site. This feature enables a dual-release op-tion for each GBpart: BsaI release allows cloning ina-destination vectors, whereas BtgZI release allowscloning in V-destination vectors. We noted that BtgZI/BsmBI assemblies are less efficient than BsaI ones. De-spite this drawback, the ability to create new TUs inboth destination vectors can save one cloning step, whichspeeds up the construction of new multigene assembliesand opens up new possibilities for automation.

We also developed a number of tools to assist usersin their engineering projects. First, we anticipated ge-netic designer needs by prearranging a number offrequently used structures. Then, we populated our in-house collection with all the elements (GBparts, GBSparts,and software tools) required to enable the use of thefrequently used structures. Finally, we assayed thefunctionality of newly developed elements using inplanta assays. In certain cases, this implied an initialstep toward part characterization. One of the hallmarksof synthetic biology is its ability to predict the behaviorof a system based on the characteristics of its constitu-tive parts. We show herein that it is possible to infer theactivity provided by a “promoter + terminator” pair

Figure 6. GoldenBraid versus GB2.0. The previous GoldenBraid version had an asymmetric assembly flow (left). GBpartsincorporated either the BsaI or the BsmBI releasable overhangs. BsaI-released GBparts were incorporated into the GoldenBraidcloning loop through level a vectors, whereas the BsmBI GBparts were used to build composite parts through the level V entrypoint. In the new GB2.0 symmetric design, the same GBparts can be incorporated into level a plasmids by a BsaI restriction/ligation reaction or into level V vectors by a mixed BsmBI/BtgZI reaction. Other differences between the previous GoldenBraidversion and GB2.0 are also listed. TA Cloning, PCR bands cloned using adenosine/timine overgangs. [See online article forcolor version of this figure.]





from the activities that each individual element displaywhen separately assayed. The differences observed be-tween the theoretical and experimental activity valuesfall within a narrow range that comes close to 0, withvery few combinations showing deviations that areslightly above 2-fold (60.3 in log values). This findingis important for engineering attempts that, as in com-plex metabolic engineering, require the combination ofmany different noncoding parts to create large meta-bolic pathways, while avoiding the introduction ofunstable repetitive regions into the genetic design. Thepromoter parts assayed herein reveal a wider range ofactivities than terminators. Nevertheless, we confirmthat the use of strong terminators like TAtHSP18.2 canpromote the promoter’s transcriptional activity, asdescribed previously (Nagaya et al., 2010). It is inter-esting that most of the observed positive deviations re-sult from the combinations involving CaMV 35S-derivedparts, suggesting a nonlinear behavior of the CaMV 35Sregulatory elements. We employed N. benthamiana tran-sient expression and the Renilla/luciferase reporter sys-tem (Grentzmann et al., 1998) as a first step toward thecharacterization of regulatory elements. This transientmethodology is simple and accurate and, therefore, fa-cilitates the analysis. A more detailed characterizationmay need to include the developmental and tissue-specific information obtained through stable planttransformation.Both GB2.0 and the GoldenBraid collections come

into being with an open-source vocation. We rein-forced this point by developing a new set of Golden-Braid destination vectors based on open-sourcepCAMBIA binary vectors (Roberts et al., 1997; Chi-Ham et al., 2012). As we see it, the intellectual com-mons Intellectual Property model is that which bestsuits the requirements for the free exchange of partsand modules in plant synthetic biology (Oye andWellhausen, 2010). Nevertheless, a number of issues,such as the Intellectual Property of individual partsand the ability to freely distribute them, need to beaddressed in a concerted manner. Undoubtedly,community effort made to create publicly availablecollections of synthetic parts will have an impact onthe progress of this discipline.Plant synthetic biology has the potential of bringing

about a significant impact on crop production. Engi-neering enhanced abiotic stress tolerance for growthin marginal lands, turning C3 plants into C4 plants(Caemmerer et al., 2012), constructing whole-organismbiosensors or sentinels (Antunes et al., 2011), engi-neering highly challenging metabolic routes (Farreet al., 2012), and combinations of these are just some ex-amples of high-impact goals within the reach of biotech-nologists. Also, it has not escaped our notice that theproposed grammar can be easily adopted by other non-plant systems as well. We believe that technologies likeGB2.0, which enable the standardization and facilitate thecharacterization and exchange of genetic parts and mod-ules, are important contributions to the achievement ofthe challenging biotechnology goals ahead.

MATERIALS AND METHODS

Strains and Growth Conditions

Escherichia coli DH5a was used for cloning. Agrobacterium tumefaciens strainGV3101 was used for transient expression and transformation experiments.Both strains were grown in Luria-Bertani medium under agitation (200 rpm)at 37°C and 28°C, respectively. Ampicillin (50 mg mL21), kanamycin (50 mgmL21), and spectinomycin (100 mg mL21) were used for E. coli selection.Rifampicin, tetracycline, and gentamicin were also used for A. tumefaciensselection at 50, 12.5, and 30 mg mL21, respectively. 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside acid (40 mg mL21) and isopropylthio-b-galactoside(0.5 mM) were used on Luria-Bertani agar plates for the white/blue selection ofclones.

Restriction-Ligation Assembly Reactions

Restriction-ligation reactions were set up as described elsewhere (Sarrion-Perdigones et al., 2011) using BsaI, BsmBI, BtgZI, or BbsI as restriction enzymes(New England Biolabs) and T4 Ligase (Promega). Reactions were set up in25- or 50-cycle digestion/ligation reactions (2 min at 37°C, 5 min at 16°C),depending on assembly complexity. One microliter of the reaction wastransformed into E. coli DH5a electrocompetent cells, and positive clones wereselected in solid medium. Plasmid DNA was extracted using the E.Z.N.A.Plasmid Mini Kit I (Omega Bio-Tek). Assemblies were confirmed by restrictionanalysis and sequencing.

GBpart Domestication

GBparts and GBpatches were obtained by PCR amplification using suitabletemplates. The Phusion High-Fidelity DNA Polymerase (ThermoScientific) wasused for amplification following the manufacturer’s protocols. Primers smallerthan 60-mers were purchased from Sigma-Aldrich. 60-mer or longer oligo-nucleotides were synthesized by IDTDNA (Coralville) by the Ultramer tech-nology. Amplified bands were purified using the QIAquick PCR PurificationKit (Qiagen) and were quantified in the Nano Drop Spectrophotometer 2000.Then, 40 ng of each amplicon and 75 ng of the domestication vector (pUPD)were mixed and incubated in a BsmBI restriction-ligation reaction. The pUPDsequence is deposited in the GBdatabase. Positive clones were selected onampicillin-, 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside acid, and iso-propylthio-b-galactoside-containing plates, and the correct assembly wasconfirmed by restriction analyses and sequencing. A description of theGBparts and GBSparts employed in this work is provided in SupplementalTable S1. The nucleotide sequences of all the GoldenBraid parts in the col-lection are deposited in the GoldenBraid database.

pDGB Construction

Two pDGB series, pDGB1 and pDGB2, were constructed. pDGB1 is basedon the pGreenII backbone (Hellens et al., 2000), and pDGB2 is based onpCAMBIA (Roberts et al., 1997). For pDGB construction, the backbone of eachbinary vector was divided into fragments (vector modules). The pDGB1backbone comprised two fragments, whereas the pDGB2 backbone was di-vided into four modules, given the presence of internal sites. To build vectormodules, each fragment was amplified by PCR in a similar procedure to thatdescribed for GBparts and was cloned into a vector domestication plasmid(pVD) using a BsaI digestion-ligation reaction. The pVD vector was derivedfrom pUPD; its sequence was deposited in GBdatabase. In addition to thebackbone modules, a number of common modules were built: eight GBcassettes(a1, a1R, a2, a2R, V1, V1R, V2, and V2R) and two fragments encoding specti-nomycin and kanamycin resistance. To assemble each pDGB, a BbsI restriction-ligation reaction was performed by combining the modules of the vector backbone,the desired GBcassette, and appropriate antibiotic resistance.

Nicotiana benthamiana Transient Transformation

For the transient expression experiments, plasmids were transferred toA. tumefaciens strain GV3101 by electroporation. Agroinfiltration was per-formed as described previously (Wieland et al., 2006). Overnight-grownbacterial cultures were pelleted and resuspended in agroinfiltration medium(10 mM MES, pH 5.6, 10 mM MgCl2, and 200 mM acetosyringone) to an optical


GoldenBraid 2.0





density at 600 nm of 0.5. Infiltrations were carried out using a needle-freesyringe in leaves 2, 3, and 4 of 4- to 5-week-old N. benthamiana plants(growing conditions: 24°C day/20°C night in a 16-h-light/8-h-dark cycle).Depending on the purpose of the experiments, leaves were harvested 3 to5 d post infiltration and examined for transgene expression.

Arabidopsis Stable Transformation

Arabidopsis (Arabidopsis thaliana) accession Columbia plants were trans-formed by the floral dip method (Clough and Bent, 1998). Seeds were steril-ized and plated on plates of Murashige and Skoog medium with 0.8% (w/v)agar and 1% (w/v) Suc (growing conditions: 24°C day/20°C night in a 16-h-light/8-h-dark cycle). Transgenic lines were selected without antibiotic resis-tance, as PDS silencing-transformed lines showed the albino phenotype.

Renilla/Luciferase Expression Assays

In order to measure the activity of Renilla/luciferase reporters (Grentzmannet al., 1998), three or four N. benthamiana leaves were agroinfiltrated followingthe above-described procedure. Leaves were harvested 3 d post infiltration.Firefly luciferase and Renilla luciferase were assayed from 100-mg leaf extractsfollowing the Dual-Glo Luciferase Assay System (Promega) standard protocoland were quantified with a GloMax 96 Microplate Luminometer (Promega).The ETA of each region was calculated in relation to the Renilla/luciferasevalues of a (01–12_17–21) reference combination (PNos_TNos), which wasarbitrarily set as 1, according to the formulae:

ETAð01� 12Þi ¼ FLuc=RLuc�ð01� 12Þi ð17� 21ÞTNos

��FLuc=RLuc

3�ð01� 12ÞPNos ð17� 21ÞTNos

�

ETAð17�21Þj ¼ FLuc=RLuc�ð01� 12ÞPNos ð17� 21Þj

��FLuc=RLuc


�;

where FLuc/RLuc [(01–12)i_(17–21)j] refers to the ratio between the fireflyluciferase activity (FLuc) of a (01–12)i:luciferase:(17–21)j construct and theRenilla luciferase activity (RLuc) of a 35S:Renilla:TNos internal standardconstruct. TTA was calculated for each cassette combination as the product ofthe individual ETA of the two regulatory regions, as follows:

TTAij ¼ ETAð01� 12Þi3ETAð17� 21ÞjFinally, the FLuc/RLuc of a number of cassette combinations was tested ex-perimentally, and the ETA of each combination (ETAij) was calculated withthe formula:

ETAij ¼ FLuc=RLuchð01� 12Þi ð17� 21Þj

i.FLuc=RLuc


�:

Glucocorticoid Receptor Inductionand Heat-Shock Treatments

Discs of 1 cm2 from agroinfiltrated leaves were harvested at 3 d post in-filtration, placed in a 350-mL solution containing 5 to 20 mM dexamethasone(Sigma-Aldrich) in 0.02% Tween 80, and incubated overnight in a growthchamber. Firefly luciferase and Renilla luciferase activities were measuredafter 24 h of treatment (6-h-light/8-h-dark, 24°C). For the heat-shock treat-ments, 1 cm2 of leaves at 3 d post infiltration was placed in 350 mL of water at37°C for 2 h. Samples were collected at 3 and 14 h after treatment.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. GB Domestication with the removal of internalrestriction sites.

Supplemental Figure S2. Combinatorial antibody engineering using theGBpatch assembly level.

Supplemental Figure S3. Theoretical and experimental transcriptional ac-tivity of different promoter/terminator combinations.

Supplemental Figure S4. Functional characterization of heat-shockpromoters.

Supplemental Figure S5. Functional characterization of transactivationconstructs.

Supplemental Figure S6. Frequently Used Structures (FUS) for the protein-protein interaction analysis.

Supplemental Figure S7. The Frequently Used Structures (FUS) for endog-enous gene silencing.

Supplemental Figure S8. Adapting Gateway (GW) technology to GB2.0.

Supplemental Table S1. GBparts used in this work.

ACKNOWLEDGMENTS

We particularly thank Dr. S. Marillonnet for his efforts in agreeing on acommon standard for MoClo and GB2.0. Many people provided us with ma-terial and knowledge, which helped us to build our GBcollection. From ourresearch center (Instituto de Biología Molecular y Celular de Plantas), Dr. M.A.Blázquez and Dr. D. Alabadí shared their knowledge on Arabidopsis pro-moters, Dr. A. Ferrando shared the BiFC vectors, Dr. C. Ferrandiz helpedwith the protein-protein interaction examples, Dr. J. Carbonell and Dr. C.Urbez helped with the amiRNA work, while Dr. J.A. Darós provided assis-tance with the fluorescent proteins and silencing suppressors. HSP70B wasprovided by Prof. A. Maule (John Innes Centre). Dr. F. Felippes and Dr. D.Wiegel (Max Planck Institute for Developmental Biology) shared detailed in-formation on the microRNA-induced gene silencing design.

Received March 15, 2013; accepted May 10, 2013; published May 13, 2013.

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GoldenBraid 2.0



GoldenBraid 2.0: A Comprehensive DNA Assembly · transfer of multigene structures to the plant...

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