DNA assembly for synthetic biology: from parts to pathways and beyond

2011 ◽  
Vol 3 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Tom Ellis ◽  
Tom Adie ◽  
Geoff S. Baldwin
Keyword(s):  
Synthetic Biology ◽  
Dna Assembly

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.

scholarly journals Erratum to: No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

2016 ◽  
Vol 10 (1) ◽  
Author(s):  
Afnan Azizi ◽  
Wilson Lam ◽  
Hilary Phenix ◽  
Lioudmila Tepliakova ◽  
Ian J. Roney ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Best Practice ◽  
Experimental Tests ◽  
Dna Assembly

scholarly journals Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

2019 ◽  
Vol 85 (16) ◽  
Author(s):  
Céline Aubry ◽  
Jean-Luc Pernodet ◽  
Sylvie Lautru
Keyword(s):  
Synthetic Biology ◽  
Anticancer Agents ◽  
Gene Clusters ◽  
Bioactive Molecules ◽  
Dna Assembly ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Dna Cloning ◽  
Specialized Metabolism ◽  
Assembly Method

ABSTRACT With the development of synthetic biology in the field of (actinobacterial) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed. We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FLP recombination target sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalyzed excision, were all confirmed. To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the BioBrick assembly method. We also used the seamless ligase chain reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain. IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example, antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacterial chromosomes. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for example, for protein production or genetic complementation.

scholarly journals No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

2015 ◽  
Vol 9 (1) ◽  
Author(s):  
Afnan Azizi ◽  
Wilson Lam ◽  
Hilary Phenix ◽  
Lioudmila Tepliakova ◽  
Ian J Roney ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Best Practice ◽  
Experimental Tests ◽  
Dna Assembly

scholarly journals Correction to “The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens”

2021 ◽  
Author(s):  
Daniel Stukenberg ◽  
Tobias Hensel ◽  
Josef Hoff ◽  
Benjamin Daniel ◽  
René Inckemann ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Assembly ◽  
Golden Gate

MIDAS: A Modular DNA Assembly System for Synthetic Biology

2018 ◽  
Vol 7 (4) ◽  
pp. 1018-1029 ◽  
Author(s):  
Craig J. van Dolleweerd ◽  
Sarah A. Kessans ◽  
Kyle C. Van de Bittner ◽  
Leyla Y. Bustamante ◽  
Rudranuj Bundela ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Assembly System ◽  
Dna Assembly

scholarly journals BASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology

2015 ◽  
Vol 4 (7) ◽  
pp. 781-787 ◽  
Author(s):  
Marko Storch ◽  
Arturo Casini ◽  
Ben Mackrow ◽  
Toni Fleming ◽  
Harry Trewhitt ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Assembly

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 Y la biología se convirtió en ingeniería: La adopción de estándares para sistemas vivos

2020 ◽  
Author(s):  
Victor De Lorenzo
Keyword(s):  
Synthetic Biology ◽  
Production Process ◽  
Industrial Production ◽  
Industrial Design ◽  
Dna Assembly ◽  
Fundamental Knowledge ◽  
Biological Objects ◽  
Activity Measurements ◽  
One Step ◽  
Design Construction

For decades, molecular biologists have been removing or inserting genes into all kinds of organisms with biotechnological intent or simply to generate fundamental knowledge. Synthetic biology (SynBio) goes one step further by incorporating conceptual frameworks from computing, electronics, and industrial design. This change makes it possible to conceive the creation of complex biological objects that were previously considered too difficult to assemble. To do this, the stages of any industrial production process must be adopted: design, construction of the components, assembly, and final manufacture. This objective requires standardisation of the physical and functional formats of the components involved, DNA assembly methods, activity measurements, and descriptive languages.

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