Seamless Assembly Research Articles

Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments

The Golden Gate method is an efficient tool for seamless assembly of multiple DNA fragments, which uses Type IIS restriction endonucleases, cleaving the DNA outside of their recognition site to release DNA parts from PCR fragments or entry clones, thus allowing the design of overhangs for ligation at will. However, the construction of the entry clones requires the use of other restriction enzyme(s) or cloning techniques and different entry vectors for the individual overhangs. Here, we present a simplified Golden Gate cloning approach termed Golden EGG. It features (1) a single entry vector with a specific cloning site to host the DNA parts; (2) a unique primer design to create the restriction enzyme recognition site to release the fragments with the overhangs at will; (3) the use of a single Type IIS enzyme for the construction of both the entry and destination clones; (4) a specific temperature profile during the digestion-ligation reaction. Our user-friendly, streamlined method retains the key attributes of the Golden Gate technique, while offering the potential to generate compatible parts with any existing Golden Gate toolkit and to be accessible to a wide user base without the need for extensive acquisition of new vectors or expensive enzymes.

Kinematic modeling and formability analysis of revolved bodies formed by origami waterbomb units based on a chain-like layer-building method

ABSTRACT Origami patterns play a critical role in the field of soft and reconfigurable robotics. The revolved body formed by waterbomb units is also widely used in robot design. The kinematic model plays a key role in understanding motion characteristics and is vital for dynamic modeling and control of robots based on origami. However, while the deformation of origami patterns is well-studied on the finite element level, the assembly of rigid origami patterns is rarely explored on the kinematic level. Therefore, we propose a chain-like assembly method for constructing revolved bodies using rigid waterbomb units, ensuring crease status unalternation, collision avoidance, axis existence, and seamless assembly. We explore the formability of waterbomb units and investigate the resulting revolved body with some layer and column numbers of waterbomb units. Results demonstrate that increasing the number of columns does not necessarily provide more space for building layers. Additionally, they reveal boundaries of the layer and column numbers for constructing the revolved body and highlight the impact of the aspect ratio and configuration of the waterbomb units on the formability of the revolved body. This method can provide insights for origami-based robot research and can be extended to model other origami patterns.

Applications of Programmable Endonucleases in Sequence- and Ligation-Independent Seamless DNA Assembly.

Programmable endonucleases, such as Cas (Clustered Regularly-Interspaced Short Repeats-associated proteins) and prokaryotic Argonaute (pAgo), depend on base pairing of the target DNA with the guide RNA or DNA to cleave DNA strands. Therefore, they are capable of recognizing and cleaving DNA sequences at virtually any arbitrary site. The present review focuses on the commonly used in vivo and in vitro recombination-based gene cloning methods and the application of programmable endonucleases in these sequence- and ligation-independent DNA assembly methods. The advantages and shortcomings of the programmable endonucleases utilized as tools for gene cloning are also discussed in this review.

HECA: The cell-centric assembly of a cell atlas

SummaryThe accumulation of massive single-cell omics data provides growing resources for building biomolecular atlases of all cells of human organs or the whole body. The true assembly of a cell atlas should be cell-centric rather than file-centric. We developed a unified informatics framework for seamless cell-centric data assembly and built the human Ensemble Cell Atlas (hECA) from scattered data. hECA v1.0 assembled 1,093,299 labeled human cells from 116 published datasets, covering 38 organs and 11 systems. We invented three new methods of atlas applications based on the cell-centric assembly: “in data” cell sorting for targeted data retrieval with customizable logic expressions, “quantitative portraiture” for multi-view representations of biological entities, and customizable reference creation for generating references for automatic annotations. Case studies on agile construction of user-defined sub-atlases and “in data” investigation of CAR-T off-targets in multiple organs showed the great potential enabled by the cell-centric ensemble atlas.

Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity.

Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (SynViP). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See'm principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering.

A Biodegradable Secondary Battery and its Biodegradation Mechanism for Eco-Friendly Energy-Storage Systems.

The production of rechargeable batteries is rapidly expanding, and there are going to be new challenges in the near future about how the potential environmental impact caused by the disposal of the large volume of the used batteries can be minimized. Herein, a novel strategy is proposed to address these concerns by applying biodegradable device technology. An eco-friendly and biodegradable sodium-ion secondary battery (SIB) is developed through extensive material screening followed by the synthesis of biodegradable electrodes and their seamless assembly with an unconventional biodegradable separator, electrolyte, and package. Each battery component decomposes in nature into non-toxic compounds or elements via hydrolysis and/or fungal degradation, with all of the biodegradation products naturally abundant and eco-friendly. Detailed biodegradation mechanisms and toxicity influence of each component on living organisms are determined. In addition, this new SIB delivers performance comparable to that of conventional non-degradable SIBs. The strategy and findings suggest a novel eco-friendly biodegradable paradigm for large-scale rechargeable battery systems.

Function block-based human-robot collaborative assembly driven by brainwaves

As an emerging communication modality, brainwaves can be used to control robots for seamless assembly, especially in noisy environments where voice recognition is not reliable or when an operator is occupied with other tasks and unable to make gestures. This paper investigates human-robot collaborative assembly based on function blocks and driven by brainwaves. Using wavelet transform, brainwaves measured by EEG sensors are converted to time-frequency images and subsequently classified by a convolutional neural network (CNN) as commands to trigger a network of function blocks for assembly actions. The effectiveness of the system is experimentally validated through an engine-assembly case study.

Targeted Disruption of Scytalone Dehydratase Gene Using Agrobacterium tumefaciens-Mediated Transformation Leads to Altered Melanin Production in Ascochyta lentis.

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete Ascochyta lentis which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in AlKewell. Two SCD genes have been identified in AlKewell, AlSCD1 and AlSCD2. Phylogenetic analysis revealed that AlSCD1 clustered with the previously characterized fungal SCDs; thus, AlSCD1 was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting AlKewell scd1::hyg transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of AlSCD1 gene did not result in a change in the virulence profile of AlKewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in A. lentis which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.

Development of a Full-Length Infectious cDNA Clone of the Grapevine Berry Inner Necrosis Virus

Grapevine berry inner necrosis virus (GINV) belongs to the genus Trichovirus in the family Betaflexiviridae. The GINV isolate LN_BETA_RS was obtained from a “Beta” grapevine (Vitis riparia × Vitis labrusca) exhibiting chlorotic mottling and ring spot in Xingcheng, Liaoning Province, China. To verify the correlation between GINV and grapevine chlorotic mottling and ring spot disease, we constructed an infectious cDNA clone of GINV isolate LN_BETA_RS using the seamless assembly approach. Applied treatments of agroinfiltration infectious cDNA confirmed systemic GINV infection of the Nicotiana occidentalis 37B by reverse transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy, exhibiting chlorotic mottling symptoms on leaves. Infectious cDNA was also transmitted to new healthy N. occidentalis plants through rub-inoculation. Moreover, the cDNA clone was agroinfiltrated into “Beta” and “Thompson Seedless” grapevine plantlets, and the inoculated grapevines exhibited leaf chlorotic mottling and ringspot during the two years of observation. GINV-inoculated “Beta” grapevines had serious leaf chlorotic mottling and ringspot symptoms on the whole plant, while relatively few symptoms were observed on the leaves of agroinoculated “Thompson Seedless” grapevines in early spring and only weak ring spot gradually appeared later in the top young leaves. Our experiments fulfilled Koch’s postulates and revealed the causative role of GINV in grapevine chlorotic mottling and ring spot disease.

Development of a Method for Simultaneous Generation of Multiple Genetic Modification in Salmonella enterica Serovar Typhimurium

To comprehensively analyze bacterial gene function, it is important to simultaneously generate multiple genetic modifications within the target gene. However, current genetic engineering approaches, which mainly use suicide vector- or λ red homologous recombination-based systems, are tedious and technically difficult to perform. Here, we developed a flexible and easy method to simultaneously construct multiple modifications at the same locus on the Salmonella enterica serovar Typhimurium chromosome. The method combines an efficient seamless assembly system in vitro, red homologous recombination in vivo, and counterselection marker sacB. To test this method, with the seamless assembly system, various modification fragments for target genes cpxR, cpxA, and acrB were rapidly and efficiently constructed in vitro. sacBKan cassettes generated via polymerase chain reaction were inserted into the target loci in the genome of Salmonella Typhimurium strain CVCC541. The resulting pKD46-containing kanamycin-resistant recombinants were selected and used as intermediate strains. Multiple target gene modifications were then carried out simultaneously via allelic exchange using various homologous recombinogenic DNA fragments to replace the sacBKan cassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 × FLAG tagging of the target genes. This method can be used in any bacterial species that supports sacB gene activity and λ red-mediated recombination, allowing in-depth functional analysis of bacterial genes.

An expanded library of orthogonal split inteins enables modular multi-peptide assemblies

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.

Scalable CVD synthesis of three-dimensional graphene from cast catalyst

Three-dimensional (3D) graphene has become an area of great interest due to its unique properties, and different synthesis routes and structures have been reported in the literature. However, the challenges of retaining high quality, scaling up and improving economic feasibility are apparent. Here we describe 3D graphene sheet (3DGS), which is a novel, electrically conductive and porous 3D graphene material that addresses some of these challenges. Due to the combination of polymer binders and a casting technique, scale up for this material is possible. The 3DGS reported in this work can be described as a seamless and freestanding assembly with low structural defects, electrical conductivity of 14.71 S/cm and reasonable mechanical integrity. This material exhibits an average density of 21.65 ± 1.49 kg/m3 and a mesoporous structure with a specific surface area of 16.5 m2/g. The Chemical Vapor Deposition (CVD) synthesis and the cast catalyst preparation method is simple and versatile in terms of tuning the material properties such as areal density and electrical conductivity. These and other properties of 3DGS enable its use in many applications, especially as binder-free, flexible electrodes and current collectors for energy storage devices and sensors.

From CT to 3D Printed Models, Serious Gaming, and Virtual Reality: Framework for Educational 3D Visualization of Complex Anatomical Spaces From Within-the Pterygopalatine Fossa.

We describe the framework for capturing the internal view of complex anatomical spaces via multiple media and haptic platforms, exemplified by realistic and conceptual representations of the pterygopalatine fossa (PPF). A realistic three-dimensional (3D) mesh of the PPF was developed by segmenting the osseous anatomy on computed tomography (CT) using Materialize InPrint. Subsequently in Autodesk 3D Studio Max, the realistic mesh was enhanced with graphically designed neurovascular anatomy and additionally a conceptual representation of the PPF with its connections and contents was created. An interactive web-compatible Adobe Flash tutorial using ActionScript was developed, allowing users to advance through a series of educational slides that contained interactive rotatable interior camera views and scrollable CT cross-sectional content, incorporating both the realistic and conceptual models. Both models were also 3D printed using polyamide material. In the realistic model, the neurovasculature was colored with water-based acrylic paint. A 3-piece modular design with embedded magnets allows for internal visualization and seamless assembly. A serious gaming environment of the conceptual PPF was also developed using Truevision3D application programming interface, where users can freely move around rooms and hallways that represent various spaces. Lastly, the realistic model was incorporated into a headset-based virtual reality environment, Surgical Theater, allowing visualization and fly-through inside and outside the model. Multiple 3D techniques for visualization of complex 3D anatomical spaces from within were described, with the necessary software and skills detailed. A rough estimate of the time and cost needed to develop these tools as well as multiple supplementary source and end result files are also made available. Educators could utilize multiple advanced delivery methods to incorporate custom digital 3D models of complex anatomical spaces understood from inside.

Seamless insert-plasmid assembly at sub-terminal homologous sequences

The engineering of fusion proteins for structural biology and protein nanotechnology often requires seamless DNA assembly with slight variations in the domain boundaries. To improve the molecular biology workflow for such projects, we evaluated the use of sub-terminal homologous sequences (HS) for co-transformation cloning and for T5 exonuclease / Phusion DNA polymerase mediated in vitro assembly. To quantify the effects of different HS-to-ends distances on cloning efficiency, we designed a blue-white-pink screening system that allowed us to easily identify positive clones (blue colonies), negative clones resulting from circular template plasmid (pink colonies) and negative colonies originating from linearized plasmids that have recircularized without an insert (white colonies). Our experiments show that both methods are feasible with HS-to-ends distances up to at least 10 base pairs. Using a combination of co-transformation cloning at sub-terminal HS and nucleotide insertions in non-annealing primer 5′-overhangs, we integrated a fusion protein into the third intracellular loop (ICL) of a G-protein-coupled receptor (GPCR) with nine different linker boundaries, using only a single plasmid linearization reaction. This molecular cloning approach is an invaluable tool for protein engineering, protein nanotechnology and synthetic biology that extends the range of applications of DNA assembly strategies.

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing β-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced β-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of β-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.

A seamless and iterative DNA assembly method named PS-Brick and its assisted metabolic engineering for threonine and 1-propanol production

BackgroundDNA assembly is an essential technique enabling metabolic engineering and synthetic biology. Combining novel DNA assembly technologies with rational metabolic engineering can facilitate the construction of microbial cell factories. Amino acids and derived biochemicals are important products in industrial biotechnology with wide application and huge markets. DNA assembly scenarios encountered in metabolic engineering for the construction of amino acid and related compound producers, such as design-build-test-learn cycles, construction of precise genetic circuits and repetitive DNA molecules, usually require for iterative, scarless and repetitive sequence assembly methods, respectively.ResultsRestriction endonuclease (RE)-assisted strategies constitute one of the major categories of DNA assembly. Here, we developed a Type IIP and IIS RE-assisted method named PS-Brick that comprehensively takes advantage of the properties of PCR fragments and REs for iterative, seamless and repetitive sequence assembly. One round of PS-Brick reaction using purified plasmids and PCR fragments was accomplished within several hours, and transformation of the resultant reaction product from this PS-Brick assembly reaction exhibited high efficiency (104–105 CFUs/µg DNA) and high accuracy (~ 90%). An application of metabolic engineering to threonine production, including the release of feedback regulation, elimination of metabolic bottlenecks, intensification of threonine export and inactivation of threonine catabolism, was stepwise resolved in E. coli by rounds of “design-build-test-learn” cycles through the iterative PS-Brick paradigm, and 45.71 g/L threonine was obtained through fed-batch fermentation. In addition to the value of the iterative character of PS-Brick for sequential strain engineering, seamless cloning enabled precise in-frame fusion for codon saturation mutagenesis and bicistronic design, and the repetitive sequence cloning ability of PS-Brick enabled construction of tandem CRISPR sgRNA arrays for genome editing. Moreover, the heterologous pathway deriving 1-propanol pathway from threonine, composed of Lactococcus lactis kivD and Saccharomyces cerevisiae ADH2, was assembled by one cycle of PS-Brick, resulting in 1.35 g/L 1-propanol in fed-batch fermentation.ConclusionsTo the best of our knowledge, the PS-Brick framework is the first RE-assisted DNA assembly method using the strengths of both Type IIP and IIS REs. In this study, PS-Brick was demonstrated to be an efficient DNA assembly method for pathway construction and genome editing and was successfully applied in design-build-test-learn (DBTL) cycles of metabolic engineering for the production of threonine and threonine-derived 1-propanol. The PS-Brick presents a valuable addition to the current toolbox of synthetic biology and metabolic engineering.

Seamless assembly of DNA parts into functional devices and higher order multi-device systems.

A new method is introduced allowing seamless assembly of independent, functionally tested, blunt-end double strand nucleic acid parts (DNA fragments not supplied in vectors such as plasmids) into more complex biological devices (e.g. protein expression vectors) and higher order multi-device systems (e.g. biochemical pathways). Individual parts include bacterial selection markers and origins of replication, promoters useful in a variety of species, transcription terminators, shuttle sequences and a variety of “N” and “C” terminal solubility/affinity protein tags. Parts are not subjected to pre-assembly manipulation with nucleic acid modifying enzymes. Instead, they are simply mixed in appropriate pre-defined combinations and concentrations and then seamlessly linked into devices employing a specialized thermostable enzyme blend. Combinatorial assembly of parts is an inherent time-saving feature of the new method, in contrast to hierarchical binary assembly (“one part at a time”) methods. This feature substantially simplifies and speeds optimization of device and system development. The versatility and functionality of the new method was shown by combinatorial assembly of parts into vector devices, one of which optimally expressed protein from a model gene. Also, a four-enzyme biosynthetic pathway system was re-created by combinatorial construction from parts and devices. Concepts discussed in this paper provide synthetic biologists, chemists and bio-engineers with improved and expanded capability to create novel biological molecules and systems.

Seamless assembly of recombinant adenoviral genomes from high-copy plasmids.

The adenoviruses are essential tools for basic research and therapeutic development. Robust methods for the generation of mutant and recombinant viruses are crucial for these diverse applications. Here we describe a simple plasmid-based method that permits highly efficient modification of the adenoviral genome and rapid production of high-titer virus stocks. The 36-kilobase genome of adenovirus serotype 5 was divided into seven tractable blocks that could be individually modified in a single step and reassembled in vitro. Because the system is composed of compact modules, modifications at different loci can be readily recombined. Viral assemblies were delivered to packaging cells by electroporation, a strategy that consistently resulted in the de novo production of 108 infectious units in 3–5 days. In principle, a similar strategy could be applied to any other adenovirus serotype or to other double-strand DNA viruses.

Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis

To investigate gene function in Bacillus anthracis, a high-efficiency cloning system is required with an increased rate of allelic exchange. Golden Gate cloning is a molecular cloning strategy allowing researchers to simultaneously and directionally assemble multiple DNA fragments to construct target plasmids using type IIs restriction enzymes and T4 DNA ligase in the same reaction system. Here, a B. anthracis S-layer protein EA1 allelic exchange vector was successfully constructed using the Golden Gate method. No new restriction sites were introduced into this knockout vector, and seamless assembly of the DNA fragments was achieved. To elevate the efficiency of homologous recombination between the allelic exchange vector and chromosomal DNA, we introduced an I-SceI site into the allelic exchange vector. The eag gene was successfully knocked out in B. anthracis using this vector. Simultaneously, the allelic exchange vector construction method was developed into a system for generating B. anthracis allelic exchange vectors. To verify the effectiveness of this system, some other allelic exchange vectors were constructed and gene replacements were performed in B. anthracis. It is speculated that this gene knockout vector construction system and high-efficiency targeted gene replacement using I-SceI endonuclease can be applied to other Bacillus spp.

AFEAP cloning: a precise and efficient method for large DNA sequence assembly

BackgroundRecent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts.MethodsThe AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids.ResultsWe characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%.ConclusionsAFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.