A Modular Cloning System for Standardized Assembly of Multigene Constructs
By E. Weber, Carola Engler, Ramona Gruetzner, Stefan Werner, S. Marillonnet. PLoS ONE, 2011
1,027 citations recorded by the source corpus.
The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/807a89263fad357dd88ef0fb28678ff310810d2c
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In Molecular Biology
- Stefan Werner
- Ramona Gruetzner
- E. Weber
- Carola Engler
- S. Marillonnet
- Daniela Paula de Toledo Thomazella
- D. Dahlbeck
- B. Staskawicz
Citations in the corpus
- A CRISPR/Cas9 toolkit for multiplex genome editing in plants (2014, methodology): “The insert or the purified PCR fragment (T1T2-PCR), together with any of the binary vectors described in this report, were used to set up restriction-ligation reactions, as described elsewhere [44], using BsaI and T4 Ligase (New England Biolabs).”
- Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system (2012, methodology): “In order to eliminate the need for construct-specific cloning strategies, we also developed a modular cloning system (MoClo) that allows any multigene construct of choice to be made by using a defined set of pre-made vectors and a defined assembly strategy.(10) Basic genetic elements such as promote”
- CRISPR-Cas9 mediated mutagenesis of a DMR6 ortholog in tomato confers broad-spectrum disease resistance (2016, methodology): “To drive the expression of both CRISPR components (gRNA and Cas9), we used the Arabidopsis U6-26 (AtU6-26) promoter and a 2 × 35S promoter, respectively, as previously described (Hwang et al. , 2013). gRNAs were assembled using the Golden Gate cloning method (Weber et al. , 2011).”