scholarly journals Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system

2017 ◽  
Author(s):  
Johannes Gantner ◽  
Theresa Ilse ◽  
Jana Ordon ◽  
Carola Kretschmer ◽  
Ramona Gruetzner ◽  
...  
Keyword(s):  
Building Blocks ◽  
Research Community ◽  
Dna Assembly ◽  
Golden Gate ◽  
Hierarchical Assembly ◽  
Cloning System ◽  
Plant Parts ◽  
Modular Cloning ◽  
Cost Efficient ◽  
Combinatorial Assembly

AbstractStandardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

scholarly journals Mobius Assembly: A Versatile Framework For Golden Gate Assembly

2017 ◽  
Author(s):  
Andreas I. Andreou ◽  
Naomi Nakayama
Keyword(s):  
Synthetic Biology ◽  
Drop Out ◽  
Dna Assembly ◽  
Dna Fragments ◽  
Golden Gate ◽  
Transcriptional Units ◽  
Combinatorial Assembly

Golden Gate Assembly is a powerful synthetic biology tool, which utilizes Type IIS enzymes for unidirectional assembly of multiple DNA fragments. The simplicity of its DNA assembly and the exchangeability of standard parts greatly facilitate the generation of combinatorial assembly libraries. Currently there are two popular Golden Gate Assembly frameworks that allow multigene augmentation (MoClo and Golden Braid); they render either high cloning capacity or vector toolkit simplicity. We have developed a new Golden Gate Assembly framework called Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. Mobius Assembly is based on a two-level approach and embraces the standard overhangs defined by MoClo and Golden Braid to confer exchangeability, but with reduced domestication requirement. Furthermore, we have implemented drop-out cassettes encoding chromogenic proteins for visible cloning screening. As proofs of concept, we have functionally assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements.

scholarly journals GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

2017 ◽  
Vol 11 (1) ◽  
Author(s):  
Roland Prielhofer ◽  
Juan J. Barrero ◽  
Stefanie Steuer ◽  
Thomas Gassler ◽  
Richard Zahrl ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Pichia Pastoris ◽  
Golden Gate ◽  
Yeast Pichia Pastoris ◽  
Cloning System ◽  
Modular Cloning

scholarly journals CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

2018 ◽  
Author(s):  
Ravendran Vasudevan ◽  
Grant A.R. Gale ◽  
Alejandra A. Schiavon ◽  
Anton Puzorjov ◽  
John Malm ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Plasmid Vector ◽  
Dna Assembly ◽  
Golden Gate ◽  
Biotechnological Applications ◽  
Genomic Information ◽  
Exponential Increase ◽  
Multigene Expression ◽  
Modular Cloning ◽  
Biology Research

ABSTRACTRecent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and exchange of parts between different labs and biological systems. However, a standardised Modular Cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential in biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. We have generated a suite of parts and acceptor vectors for making i) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and ii) transient multigene expression and repression systems using known and novel replicative vectors. We have tested and compared the CyanoGate system in the established model cyanobacteriumSynechocystissp. PCC 6803 and the more recently described fast-growing strainSynechococcus elongatusUTEX 2973. The system is publicly available and can be readily expanded to accommodate other standardised MoClo parts.

scholarly journals Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

2021 ◽  
Author(s):  
Vivianne J Goosens ◽  
Kenneth T Walker ◽  
Silvia M Aragon ◽  
Amritpal Singh ◽  
Vivek R Senthivel ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Protein Secretion ◽  
Functional Expression ◽  
Dna Assembly ◽  
Cloning System ◽  
Pure Cellulose ◽  
Modular Cloning ◽  
High Yields ◽  
Short Time ◽  
Cellulose Materials

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

scholarly journals Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197185 ◽  
Author(s):  
Johannes Gantner ◽  
Jana Ordon ◽  
Theresa Ilse ◽  
Carola Kretschmer ◽  
Ramona Gruetzner ◽  
...  
Keyword(s):  
Cloning System ◽  
Plant Parts ◽  
Modular Cloning

scholarly journals Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Copy Number ◽  
Life Science ◽  
Insertion Site ◽  
Golden Gate ◽  
Common Elements ◽  
Modern Life ◽  
Specific Host ◽  
Modular Cloning ◽  
Golden Gate Cloning ◽  
Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

scholarly journals Dimerization and oligomerization of DNA-assembled building blocks for controlled multi-motion in high-order architectures

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ling Xin ◽  
Xiaoyang Duan ◽  
Na Liu
Keyword(s):  
Building Block ◽  
Degrees Of Freedom ◽  
Building Blocks ◽  
Single Type ◽  
Hierarchical Assembly ◽  
Optical Responses ◽  
Self Association ◽  
Living Organisms ◽  
Order Structures ◽  
Chiral System

AbstractIn living organisms, proteins are organized prevalently through a self-association mechanism to form dimers and oligomers, which often confer new functions at the intermolecular interfaces. Despite the progress on DNA-assembled artificial systems, endeavors have been largely paid to achieve monomeric nanostructures that mimic motor proteins for a single type of motion. Here, we demonstrate a DNA-assembled building block with rotary and walking modules, which can introduce new motion through dimerization and oligomerization. The building block is a chiral system, comprising two interacting gold nanorods to perform rotation and walking, respectively. Through dimerization, two building blocks can form a dimer to yield coordinated sliding. Further oligomerization leads to higher-order structures, containing alternating rotation and sliding dimer interfaces to impose structural twisting. Our hierarchical assembly scheme offers a design blueprint to construct DNA-assembled advanced architectures with high degrees of freedom to tailor the optical responses and regulate multi-motion on the nanoscale.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

DNA-based self-assembly of nanostructures

2017 ◽  
Author(s):  
Joshua D. Carter ◽  
Chenxiang Lin ◽  
Yan Liu ◽  
Hao Yan ◽  
Thomas H. LaBean
Keyword(s):  
Self Assembly ◽  
Materials Science ◽  
Directed Assembly ◽  
Building Blocks ◽  
Hierarchical Assembly ◽  
Synthetic Dna ◽  
Nanoscale Manufacturing ◽  
Covalently Linked ◽  
Design Construction ◽  
Proteins And Peptides

This article examines the DNA-based self-assembly of nanostructures. It first reviews the development of DNA self-assembly and DNA-directed assembly, focusing on the main strategies and building blocks available in the modern molecular construction toolbox, including the design, construction, and analysis of nanostructures composed entirely of synthetic DNA, as well as origami nanostructures formed from a mixture of synthetic and biological DNA. In particular, it considers the stepwise covalent synthesis of DNA nanomaterials, unmediated assembly of DNA nanomaterials, hierarchical assembly, nucleated assembly, and algorithmic assembly. It then discusses DNA-directed assembly of heteromaterials such as proteins and peptides, gold nanoparticles, and multicomponent nanostructures. It also describes the use of complementary DNA cohesion as 'smart glue' for bringing together covalently linked functional groups, biomolecules, and nanomaterials. Finally, it evaluates the potential future of DNA-based self-assembly for nanoscale manufacturing for applications in medicine, electronics, photonics, and materials science.

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