GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Structure of the LacZ cassettes in the GoldenBraid system.

<p>GB plasmid set comprises four destination plasmids (pDGBs), two of them act as destination plasmids for level α assembly and the remaining two function as destination plasmids for level Ω. All pDGB vectors incorporate a LacZ selection cassette flanked by four Type IIs restriction sites (BsaI, BsmBI), but positioned in inverted positions and orientations. To facilitate the visualization of the design, we assigned each 4 bp cleavage sequence a different label: those produced by BsaI digestion are labeled with squares and named with Arabic numbers (1,2,3), whereas BsmBI 4 bp cleavage sites are encircled and named with capital letters (A,B,C).</p

The mechanism of GoldenBraid system.

<p>(A) Standard parts as promoters (PR), coding sequences (CDS) and terminators (TM), flanked by fixed BsaI cleavage sites (represented as Arabic and Latin boxed numbers) are ordinarily assembled using level α plasmids (pDGBA12C or pDGBC12B). As a result of multipartite assembly, BsaI recognition sites disappear and the resulting boundary is not cleavable anymore (represented as a crossed label). Nevertheless, the newly assembled transcriptional unit (TU1, represented for simplification as an arrow) remains flanked by BsmBI cleavable sites (represented as encircled capital letters). (B) Two transcriptional units assembled in complementary α plasmids can be reused as entry vectors (pEGB) for a subsequent level Ω binary assembly, provided that they share a BsmBI sticky end (labeled as encircled C). Similarly, constructs assembled using opposite Ω plasmids can be reused as entry vectors for a subsequent level α binary assembly, provided that they share a BsaI sticky end (labeled as squared 3). Level α and level Ω can alternate indefinitely creating increasingly complex structures, as depicted by the arrows closing the double loop. Encircled K and S represent KanR and SpecR respectively. (C) Representation of the four “twister” plasmids that can be eventually used to assist GoldenBraid cloning design. SF is a 150 bp stuffer fragment containing an intergenic region from <i>Solanum.</i></p

Comparison of the topology of MoClo and GoldenBraid.

<p>(A) Hierarchical topology of MoClo assembly. Level 0 hosts the flexible assembly of subparts into basic parts, allowing also part domestication. Level 1 hosts multipartite assembly of basic parts into transcriptional units. Level 2-1 hosts multipartite assembly of transcriptional units, yielding a non-reusable structure. Alternatively, level 1 can be branched into level 2-1i (intermediate) by adding an end-linker, yielding an open structure (albeit non functional), which can host new transcriptional units (level 2-2). Successive intermediate levels ensure the indefinite structure of the cloning system. (B) Double loop topology of GoldenBraid. Level-α plasmids host the multipartite assembly of basic parts into transcriptional units. Two level-α transcriptional units can be assembled together yielding two alternative level-Ω constructs, which themselves can be assembled into level-α constructs. The overall structure is a double iterative loop that ensures the indefinite growth of the assembly system.</p

Multigenic constructs for plant biology.

<p>(A) GoldenBraid cloning path for the assembling of YFP, p19, BFP and DsRED transcriptional units in a single T-DNA. (B) Spatial expression patterns of BFP, YFP and DsRed in <i>N. benthamiana</i> leaves agroinfiltrated with pEGB_A-YFP-P19-BFP-DsRed-C- (left captures, 1, 2 and 3) or with a mixture of the individual devices pEGB_A-YFP-C, pEGB_C-p19-B, pEGB_A-BFP-C and pEGB_C-DsRed-B (right captures 4, 5 and 6). (C) GoldenBraid cloning strategy followed in the assembly of different IgA isotypes. Multipartite assembly involved the combination of different basic parts each occupying a fixed position in the assembly (P1-P5). Individual antibody chains were assembled in pDGB_C12B plasmid to yield four IgA isotypes. 35S is CaMV35S promoter; SP, pectate lyase signal peptide; CHα1 and CHα2, are heavy chain constant domains; Tnos, is nopaline synthase terminator; Cλ and Cκ, are light chain constant domains; VH and VL are heavy and light variable regions of an antibody against rotavirus VP8* peptide. Promoter and terminator pieces were flanked by the same 4 nucleotide extensions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021622#pone-0021622-g001" target="_blank">Fig 1</a>. Signal peptides incorporated a GATG extension at its 5′ end, whereas constant antibody regions ended in TGAG extensions to match terminators. The remaining boundaries were designed to produce benign junctions within coding sequences. (D) Western Blot analysis of IgA transient expression in <i>Nicotiana benthamiana</i>. Leaves were infiltrated with the four previous combinations. Samples were resolved under either reducing (left) or non-reducing (right) conditions and decorated using anti-heavy chain antibody, anti- λ light chain antibody or anti- κ light chain antibody. HS lane contains control human serum. (E) End-point antigen-ELISA tittering of four IgA combinations tested by transient expression in <i>Nicotiana benthamiana</i> leaves. All samples were tittered against VP8* or against BSA and compared with equivalent samples derived from wild type leaves (WT). (F) GoldenBraid strategy for the assembly of two alternative 5-gene T-DNA constructs. (G) PvuI digestion of one colony of each final constructs pDGB_A-KanR-IgHα1-Igλ-Barnase-Rosea-C (lane I) and pDGB_A-KanR-IgHα1-Igλ-Barnase-DsRed-C (lane II). Asterisks highlight those GB-assembled transcriptional units that were reused in the assembly of new multigenic structures.</p

Part standardization and multipartite assembly of single devices.

<p>PCR products of entry plasmids (pE) containing basic parts such as promoters (PR), coding sequences (CDS) and terminators (TM) are flanked by fixed convergent BsaI recognition-cleavage sites. To facilitate the visualization of the design, we assigned each 4 bp cleavage sequence a different label: those produced by BsaI digestion are labeled with Arabic and Latin numbers (1,2,3, III, IV, etc). In assembling a single device, constituent parts (pEs) are incubated together with a destination plasmid (pD) containing a LacZ cassette flanked by BsaI sites in divergent orientation. As a result, an expression plasmid (pEx) is created where all BsaI recognition sites have disappeared<b>.</b> Encircled letters represent antibiotic resistance genes: A for AmpR, and K for KanR.</p

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data