Part Of Library Research Articles

In- & Out-Cloning: plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly.

Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on 4-nt overhangs generated by commonly used Type IIS enzymes. However, reduction to 3-nt overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we term as In-Cloning. In-Cloning is downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnet's group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow called Out-Cloning to rapidly generate Golden Gate acceptor plasmids. Out-Cloning uses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.

GOLDBAR: A Framework for Combinatorial Biological Design.

With the rise of new DNA part libraries and technologies for assembling DNA, synthetic biologists are increasingly constructing and screening combinatorial libraries to optimize their biological designs. As combinatorial libraries are used to generate data on design performance, new rules for composing biological designs will emerge. Most formal frameworks for combinatorial design, however, do not yet support formal comparison of design composition, which is needed to facilitate automated analysis and machine learning in massive biological design spaces. To address this need, we introduce a combinatorial design framework called GOLDBAR. Compared with existing frameworks, GOLDBAR enables synthetic biologists to intersect and merge the rules for entire classes of biological designs to extract common design motifs and infer new ones. Here, we demonstrate the application of GOLDBAR to refine/validate design spaces for TetR-homologue transcriptional logic circuits, verify the assembly of a partial nif gene cluster, and infer novel gene clusters for the biosynthesis of rebeccamycin. We also discuss how GOLDBAR could be used to facilitate grammar-based machine learning in synthetic biology.

A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae.

Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.

차량 서스펜션의 최적 설계에서 사양관리와 부품 공용화를 위한 CAD 템플릿 및 부품 라이브러리 구축

차량 서스펜션의 최적 설계에서 사양관리와 부품 공용화를 위한 CAD 템플릿 및 부품 라이브러리 구축

Genetic Parts and Enabling Tools for Biocircuit Design.

Synthetic biology aims to engineer biological systems for customized tasks through the bottom-up assembly of fundamental building blocks, which requires high-quality libraries of reliable, modular, and standardized genetic parts. To establish sets of parts that work well together, synthetic biologists created standardized part libraries in which every component is analyzed in the same metrics and context. Here we present a state-of-the-art review of the currently available part libraries for designing biocircuits and their gene expression regulation paradigms at transcriptional, translational, and post-translational levels in Escherichia coli. We discuss the necessary facets to integrate these parts into complex devices and systems along with the current efforts to catalogue and standardize measurement data. To better display the range of available parts and to facilitate part selection in synthetic biology workflows, we established biopartsDB, a curated database of well-characterized and useful genetic part and device libraries with detailed quantitative data validated by the published literature.

Synergistical Mechanical Design and Function Integration for Insect-Scale On-Demand Configurable Multifunctional Soft Magnetic Robots.

Meso- or micro-scale(or insect-scale) robots that are capable of realizing flexible locomotion and/or carrying on complex tasks in a remotely controllable manner hold great promise in diverse fields, such as biomedical applications, unknown environment exploration, in situ operation in confined spaces, and so on. However, the existing design and implementation approaches for such multifunctional, on-demand configurable insect-scale robots are often focusing on their actuation or locomotion, while matched design and implementation with synergistic actuation and function modules under large deformation targeting varying task/target demands are rarely investigated. In this study, through systematical investigations on synergistical mechanical design and function integration, we developed a matched design and implementation method for constructing multifunctional, on-demand configurable insect-scale soft magnetic robots. Based on such a method, we report a simple approach to construct soft magnetic robots by assembling various modules from the standard part library together. Moreover, diverse soft magnetic robots with desirable motion and function can be (re)configured. Finally, we demonstrated (re)configurable soft magnetic robots shifting into different modes to adapt and respond to varying scenarios. The customizable physical realization of complex soft robots with desirable actuation and diverse functions can pave a new way for constructing more sophisticated insect-scale soft machines that can be applied to practical applications soon.

A MoClo-Compatible Toolbox of ECF Sigma Factor-Based Regulatory Switches for Proteobacterial Chassis.

The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components. Previously, extracytoplasmic function sigma factors (ECFs), a diverse group of alternative sigma factors with distinct promoter specificities, were shown to have great potential as context-independent regulators, but so far, they have only been used in a few model species. Here, we show that the alphaproteobacterium Sinorhizobium meliloti, which has been proposed as a plant-associated bacterial chassis for synthetic biology, has a similar phylogenetic ECF acceptance range as the gammaproteobacterium Escherichia coli. A common set of orthogonal ECF-based regulators that can be used in both bacterial hosts was identified and used to create 2-step delay circuits. The genetic circuits were implemented in single copy in E. coli by chromosomal integration using an established method that utilizes bacteriophage integrases. In S. meliloti, we demonstrated the usability of single-copy pABC plasmids as equivalent carriers of the synthetic circuits. The circuits were either implemented on a single pABC or modularly distributed on 3 such plasmids. In addition, we provide a toolbox containing pABC plasmids compatible with the Golden Gate (MoClo) cloning standard and a library of basic parts that enable the construction of ECF-based circuits in S. meliloti and in E. coli. This work contributes to building a context-independent and species-overarching ECF-based toolbox for synthetic biology applications.

Intelligent Simulation Design and Application of Garment Digitization Based on Virtual Image Recognition Technology

Abstract Realizing the digital design of clothing is an effective means to improve the design efficiency and effect, for this reason, this paper designs a virtual simulation design assistance system based on virtual image recognition technology. Wavelet Fourier descriptor and LDA technology are utilized to extract features and decrease the dimensionality of the clothing style image, while the extreme learning machine is utilized as a classifier to categorize the clothing style image. Based on the classification results, a virtual simulation model of the garment is constructed and stored in the parts library for the convenience of designers to call and design at any time to complete the construction of the design assistance system. The application of this paper’s image recognition method results in an overall style recognition rate of 94.8%, an average recognition time of 3.2 milliseconds for each sample, and an average Fourier wavelet descriptor classification accuracy of 97.5%. The size error is controlled within ±2.5%, and the mean customer satisfaction scores for all the finished sweaters are higher than 2. This paper proposes a style map recognition method that achieves high accuracy, reduces time consumption, produces finished products with minimal size errors, and increases customer satisfaction with the designed styles.

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

DTV-CNN: Neural network based on depth and thickness views for efficient 3D shape classification

Fast and effective algorithms for deep learning on 3D shapes are keys to innovate mechanical and electronic engineering design workflow. In this paper, an efficient 3D shape to 2D images projection algorithm and a shallow 2.5D convolutional neural network architecture is proposed. A smaller convolutional neural network (CNN) model is achieved by information enrichment at the preprocessing stage, i.e. 3D geometry is compressed into 2D “thickness view” and “depth view”. Fusing the depth view and thickness view (DTV) from the same projection view into a dual-channel grayscale image, can improve information locality for geometry and topology feature extraction. This approach bridges the gap between mature image deep learning technologies to the applications of 3D shape. Enhanced by several essential scalar geometry properties and only 3 projection views, a mixed CNN and multiple linear parameter (MLP) neural network model achives a validation accuracy of 92 % for ModelNet10 mesh-based dataset, while the training time is one order of magnitude less than the original multi-view CNN approach. This study also creates new 3D shape datasets from 2 open source CAD projects. Higher validation accuracy is obtained for realistic CAD datasets, i.e. 97 % for FreeCAD's mechanical part library and 95 % for KiCAD electronic part library. The training cost reduces to tens of minutes on a laptop CPU, given the smaller input data size and shallow neural network design. It is expected that this approach can be adapted for other machine learning scenarios involved in CAD geometry.

Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, β-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

From qualitative to quantitative: the state of the art and challenges for plant synthetic biology

The flourishing plant science promotes the exploding number of data and the expansion of toolkits. Plant synthetic biology is still in its early stages and requires more quantitative and predictable study. Despite the challenges, some pioneering examples have been successfully demonstrated in model plants.BackgroundsAs an increasing number of synthetic switches and circuits have been created for plant systems and of synthetic products produced in plant chassis, plant synthetic biology is taking a strong foothold in agriculture and medicine. The ever‐exploding data has also promoted the expansion of toolkits in this field. Genetic parts libraries and quantitative characterization approaches have been developed. However, plant synthetic biology is still in its infancy. The considerations for selecting biological parts to design and construct genetic circuits with predictable functions remain desired.ResultsIn this article, we review the current biotechnological progresses in field of plant synthetic biology. Assembly standardization and quantitative approaches of genetic parts and genetic circuits are discussed. We also highlight the main challenges in the iterative cycles of design‐build‐test‐learn for introducing novel traits into plants.ConclusionPlant synthetic biology promises to provide important solutions to many issues in agricultural production, human health care, and environmental sustainability. However, tremendous challenges exist in this field. For example, the quantitative characterization of genetic parts is limited; the orthogonality and the transfer functions of circuits are unpredictable; and also, the mathematical modeling‐assisted circuits design still needs to improve predictability and reliability. These challenges are expected to be resolved in the near future as interests in this field are intensifying.

A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria

A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.

An extensible vector toolkit and parts library for advanced engineering of plant genomes.

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

Using CIVT-SELEX to Select Aptamers as Genetic Parts to Regulate Gene Circuits in a Cell-Free System.

The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor-operator pairs. Researchers have tried to use aptamer-ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer-thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation.

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

Incorporating Metal Precursors towards a Library of High-resolution Metal Parts by Stereolithography

Additive manufacturing (AM) of three-dimensional (3D) metals with intricate fine features and complex geometry at the macro- and micro- scale for energy generation and storage has attracted great interest recently. Fabrication of metals using state-of-art powder-based AM technology with high resolution and tunable microstructure remains a challenge. In this work, a new method for achieving sophisticated metallic structure via the digital light process (DLP) of metal precursors containing photo-resin followed by thermal heating and chemical reduction process is presented. Thermal decomposition of 3D-printed metal compound and chemical reduction makes it promising to synthesize a morphology-tunable and geometrically complex metal, alloy, metal oxide, or multi-material component. Two potential applications of 3D-printed copper using this method are demonstrated in the electrochemical field as oxygen evolution reaction (OER) catalyst support and current collector. NiFe hydroxide@3D-printed copper can drive a 100 mA/cm2 stably with a low energy barrier in an alkaline solution. Galvanostatic cycling profile of DLP Copper/electrolyte/Li at current densities of 2 mA/cm2, 4 mA/cm2 and 10 mA/cm2 can be conducted stably for a capacity of 20 mA·h/cm2. This work is the first to realize metal-based printing with ultra-high resolution thorough photopolymerization.

Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides

Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.

BioCRNpyler: Compiling chemical reaction networks from biomolecular parts in diverse contexts.

Biochemical interactions in systems and synthetic biology are often modeled with chemical reaction networks (CRNs). CRNs provide a principled modeling environment capable of expressing a huge range of biochemical processes. In this paper, we present a software toolbox, written in Python, that compiles high-level design specifications represented using a modular library of biochemical parts, mechanisms, and contexts to CRN implementations. This compilation process offers four advantages. First, the building of the actual CRN representation is automatic and outputs Systems Biology Markup Language (SBML) models compatible with numerous simulators. Second, a library of modular biochemical components allows for different architectures and implementations of biochemical circuits to be represented succinctly with design choices propagated throughout the underlying CRN automatically. This prevents the often occurring mismatch between high-level designs and model dynamics. Third, high-level design specification can be embedded into diverse biomolecular environments, such as cell-free extracts and in vivo milieus. Finally, our software toolbox has a parameter database, which allows users to rapidly prototype large models using very few parameters which can be customized later. By using BioCRNpyler, users ranging from expert modelers to novice script-writers can easily build, manage, and explore sophisticated biochemical models using diverse biochemical implementations, environments, and modeling assumptions.

Towards ubiquitous data access service of 3D part library in cloud scheme with host independence

Most current part libraries are developed and deployed in specific usage scenario or CAD systems, which inevitably brings obstacles in information sharing and exchanging of parts. To reduce repeated development and provide a uniform platform for designers in different sites, a 3D part library in cloud scheme is presented in this paper. Host programs with predetermined specification can access the part data through an adapter according to their customized requests with uniform interfaces, which constructs a ubiquitous service. To realize host independence, the part models are created in a modeler with native ACIS, and then, they are converted into 3D files in different formats for practical needs; finally, these models are imported into CAD systems or other platforms in real designs. The framework is composed of three components, namely, as PLS (part library service) provider, PLS adapters, and PLS hosts. PLS provider is the kernel of 3D data access service in cloud scheme, while PLS adapters serve as the bridges that connect PLS provider and hosts, and the PLS can be grafted on various applications including current mainstream CAD systems as an embedded module or run on the websites or even mobile terminals. The PLS provider is deployed and maintained on cloud, and designers can acquire remote part information within a local ongoing project. In the detailed construction of this part library, diversiform knowledge in part parameters and structures is implanted to define the geometry and rule constraints for the 3D modeling, with which the backstage has the ability of conveniently editing the information in the part library for better upgrade and contrapuntally services. This concept has been implemented within a PaaS framework to provide the ubiquitous 3D part data accessing route, which has been successfully applied in a large number of enterprises, and accumulates considerable practical cases.