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 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 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

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 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 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 Universal loop assembly: open, efficient and cross-kingdom DNA fabrication

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Bernardo Pollak ◽  
Tamara Matute ◽  
Isaac Nuñez ◽  
Ariel Cerda ◽  
Constanza Lopez ◽  
...  
Keyword(s):  
High Efficiency ◽  
Final Host ◽  
Expression Vectors ◽  
Assembly Systems ◽  
Dna Assembly ◽  
Golden Gate ◽  
Proof Of Concept ◽  
Biological Engineering ◽  
Dna Libraries ◽  
Multigene Expression

Abstract Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a ‘common syntax’ that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe ‘universal Loop’ (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.

scholarly journals 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

scholarly journals BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

2017 ◽  
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.

scholarly journals Producing molecular biology reagents without purification

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252507
Author(s):  
Sanchita Bhadra ◽  
Vylan Nguyen ◽  
Jose-Angel Torres ◽  
Shaunak Kar ◽  
Stéphane Fadanka ◽  
...  
Keyword(s):  
United Kingdom ◽  
Synthetic Biology ◽  
Molecular Biology ◽  
Reverse Transcription ◽  
Isothermal Amplification ◽  
Dna Assembly ◽  
Golden Gate ◽  
Local Production ◽  
The United Kingdom ◽  
Simplified Method

We recently developed ‘cellular’ reagents–lyophilized bacteria overexpressing proteins of interest–that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

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