dna assembly
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Nano Today ◽  
2022 ◽  
Vol 42 ◽  
pp. 101352
Author(s):  
Chi Yao ◽  
Jianpu Tang ◽  
Chenxu Zhu ◽  
Sen Yang ◽  
Han Tang ◽  
...  

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.


2022 ◽  
Author(s):  
Matthew C Haines ◽  
Benedict Carling ◽  
James Marshall ◽  
Marko Storch ◽  
Paul C Freemont

Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore design spaces and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use, while enabling simple hierarchical assemblies through an idempotent format. These attributes facilitate various applications including pathway engineering, ribosome binding site tuning, fusion protein synthesis and multiplex gRNA expression. In this work we present basicsynbio, an open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio). With basicsynbio, users can access commonly used BASIC parts and linkers while robustly designing new parts and assemblies with exception handling for common design errors. Furthermore, users can export sequence data and create build instructions for manual or automated workflows. The generation of build instructions relies on the BasicBuild Open Standard which is easily parsed for bespoke workflows and is serialised in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio by assembling a collection of 30 BASIC-compatible vectors using various sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA collection encompasses plasmids containing six antibiotic resistance markers and five origins of replication from different compatibility groups, including a temperature-sensitive variant. We deposit the collection on Addgene under an OpenMTA agreement, making them available. Furthermore, these sequences are accessible from within the basicsynbio application programming interface along with other collections of parts and linkers, providing an ideal environment to design BASIC DNA assemblies for bioengineering applications.


2022 ◽  
Author(s):  
Behnam Enghiad ◽  
Pu Xue ◽  
Nilmani Singh ◽  
Aashutosh Girish Boob ◽  
Chengyou Shi ◽  
...  

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high-throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments within 3 days. PlasmidMaker should greatly expand the potential of synthetic biology.


2021 ◽  
Author(s):  
Julie Vanderstraeten ◽  
Maria João Maurício da Fonseca ◽  
Philippe De Groote ◽  
Dennis Grimon ◽  
Hans Gerstmans ◽  
...  

Abstract Background: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. Results: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. Conclusion: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.


2021 ◽  
Vol 194 ◽  
pp. 113618
Author(s):  
Xiao-Jing Zhai ◽  
Qiong-Lin Wang ◽  
Hui-Fang Cui ◽  
Xiaojie Song ◽  
Qi-Yan Lv ◽  
...  

Author(s):  
Na Ni ◽  
Fang Deng ◽  
Fang He ◽  
Hao Wang ◽  
Deyao Shi ◽  
...  

Author(s):  
Daniel Stukenberg ◽  
Tobias Hensel ◽  
Josef Hoff ◽  
Benjamin Daniel ◽  
René Inckemann ◽  
...  

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