Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

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.

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

10.1101/237768 ◽

2017 ◽

Cited By ~ 1

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.

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BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

10.1101/185108 ◽

2017 ◽

Cited By ~ 1

Author(s):

Niels Wicke ◽

David Radford ◽

Valeria Verrone ◽

Anil Wipat ◽

Christopher E. French

Keyword(s):

Synthetic Biology ◽

Cellular Differentiation ◽

Genetic Manipulation ◽

Surface Display ◽

Dna Assembly ◽

Golden Gate ◽

E Coli ◽

Production Platform ◽

Bacterial Model ◽

Golden Gate Cloning

AbstractBacillus subtilisis a valuable industrial production platform for proteins, a bacterial model for cellular differentiation and its endospores have been proposed as a vehicle for vaccine delivery. As suchB. subtilisis a major synthetic biology chassis but, unlikeEscherichia coli, lacks a standardized toolbox for genetic manipulation. EcoFlex is a versatile modular DNA assembly toolkit forE. colisynthetic biology based on Golden Gate cloning. Here we introduce BacilloFlex, an extension of the EcoFlex assembly standard toB. subtilis. Transcription units flanked by sequences homologous to loci in theB. subtilisgenome were rapidly assembled by the EcoFlex standard and subsequently chromosomally integrated. At present, BacilloFlex includes a range of multi-functionalB. subtilisspecific parts with applications including metabolic engineering, biosensors and spore surface display. We hope this work will form the foundation of a widely adopted cloning standard forB. subtilisfacilitating collaboration and the sharing of parts.

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

10.1101/426700 ◽

2018 ◽

Cited By ~ 2

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.

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Combinatorial DNA Assembly Using Golden Gate Cloning

Synthetic Biology - Methods in Molecular Biology ◽

10.1007/978-1-62703-625-2_12 ◽

2013 ◽

pp. 141-156 ◽

Cited By ~ 25

Author(s):

Carola Engler ◽

Sylvestre Marillonnet

Keyword(s):

Dna Assembly ◽

Golden Gate ◽

Golden Gate Cloning

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Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

10.1101/799585 ◽

2019 ◽

Cited By ~ 2

Author(s):

Marcos Valenzuela-Ortega ◽

Christopher French

Keyword(s):

Synthetic Biology ◽

Golden Gate ◽

Flexible Design ◽

Test Cycle ◽

Design Build ◽

Modular Cloning ◽

Multiple Cloning Sites ◽

Golden Gate Cloning

AbstractComplex multi-gene plasmids can be built from basic DNA parts in a reliable and automation friendly way using modular cloning standards, based on Golden Gate cloning. However, each toolkit or standard is limited to one or a few different vectors, which has led to an overabundance of toolkits with varying degrees of compatibility. Here, we present the Joint Universal Modular Plasmids (JUMP), a vector design that overcomes the limitations of current toolkits by expanding the paradigm of modular cloning: all vectors can be modified using modular cloning in an orthogonal way using multiple cloning sites. This allows researchers to introduce any feature into any JUMP vector and simplifies the Design-Build-Test cycle of synthetic biology. JUMP vectors are compatible with PhytoBrick basic parts, BioBricks and the Registry of Standard Biological Parts, and the Standard European Vector Architecture (SEVA). Due to their flexible design, JUMP vectors have the potential to be a universal platform for synthetic biology regardless of host and application. A collection of JUMP backbones and microbial PhytoBrick basic parts are available for distribution.

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

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 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.

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A modular cloning toolkit for genome editing in plants

10.1101/738021 ◽

2019 ◽

Cited By ~ 2

Author(s):

Florian Hahn ◽

Andrey Korolev ◽

Laura Sanjurjo Loures ◽

Vladimir Nekrasov

Keyword(s):

Genome Editing ◽

Higher Order ◽

Plant Science ◽

Golden Gate ◽

Coding Sequences ◽

Guide Rna ◽

Guide Rnas ◽

Science Community ◽

Modular Cloning ◽

Multiple Expression

AbstractBackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

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