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A modular cloning system for standardized assembly of multigene constructs.
Literature Information
| DOI | 10.1371/journal.pone.0016765 |
|---|---|
| PMID | 21364738 |
| Journal | PloS one |
| Impact Factor | 2.6 |
| JCR Quartile | Q2 |
| Publication Year | 2011 |
| Times Cited | 588 |
| Keywords | modular cloning system, multigene constructs, synthetic biology |
| Literature Type | Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't |
| ISSN | 1932-6203 |
| Pages | e16765 |
| Issue | 6(2) |
| Authors | Ernst Weber, Carola Engler, Ramona Gruetzner, Stefan Werner, Sylvestre Marillonnet |
TL;DR
This study introduces a hierarchical modular cloning (MoClo) system that enables efficient assembly of complex eukaryotic multigene constructs using type IIS restriction enzymes, significantly advancing the capabilities of synthetic biology. By constructing a 33 kb DNA molecule with 11 transcription units from 44 basic modules in just three steps, this innovation holds great potential for applications in gene stacking and metabolic engineering, addressing a major limitation in current methods.
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modular cloning system · multigene constructs · synthetic biology
Abstract
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.
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Primary Questions Addressed
- What are the potential applications of the modular cloning system in synthetic biology beyond gene stacking and metabolic engineering?
- How does the efficiency of this modular cloning system compare to traditional methods of multigene construct assembly?
- What specific challenges in the assembly of multigene constructs does this hierarchical modular cloning system address?
- In what ways can the automated aspects of the modular cloning system enhance the speed and accuracy of synthetic biology projects?
- What types of regulatory and coding sequences are included in the basic modules of this cloning system, and how do they influence the functionality of the constructs?
Key Findings
Research Background and Objectives
The field of synthetic biology aims to revolutionize biotechnology by engineering organisms with novel phenotypes for applications in medicine, agriculture, and industry. A significant challenge is the efficient assembly of complex DNA constructs containing multiple genetic elements. This study introduces a modular cloning system that enables the high-efficiency creation of eukaryotic multigene constructs from standardized genetic modules.
Main Methods/Materials/Experimental Design
The proposed modular cloning (MoClo) system utilizes type IIS restriction enzymes for the assembly of DNA fragments in a defined order. The system consists of hierarchical levels of modules:
- Level 0 Modules: Basic elements such as promoters, untranslated regions, signal peptides, coding sequences, and terminators.
- Level 1 Modules: Assembled transcription units created from level 0 modules.
- Level 2 Constructs: Multigene constructs formed by combining multiple level 1 modules.
The assembly process is performed in a single reaction, enhancing efficiency and allowing for automation. The cloning steps are summarized in the following Mermaid flowchart:
Key Results and Findings
- A 33 kb DNA construct containing 11 transcription units was assembled from 44 individual modules in just three cloning steps.
- The cloning efficiency was high, with up to 95-100% of colonies containing the expected constructs.
- The system allows for the incorporation of multiple transcription units in a single reaction, demonstrating versatility and efficiency in constructing complex genetic architectures.
Main Conclusions/Significance/Innovativeness
The MoClo system provides a standardized and efficient method for assembling multigene constructs, addressing limitations of previous cloning methods. Its modular nature allows for the reuse of validated genetic elements, facilitating collaboration and sharing within the scientific community. The ability to create construct libraries will be particularly beneficial for optimizing metabolic pathways in synthetic biology.
Research Limitations and Future Directions
While the MoClo system is efficient, its capacity is limited by the size of constructs that can be transformed into standard bacterial hosts. Future research may focus on:
- Expanding the number of transcription units that can be assembled in a single construct.
- Enhancing automation in the cloning process to further streamline genetic engineering workflows.
- Developing new modules and standards to improve the flexibility and utility of the system in diverse applications.
Summary Table of Key Components
| Component | Description |
|---|---|
| Level 0 Modules | Basic genetic elements (promoters, CDS, etc.) |
| Level 1 Modules | Assembled transcription units from Level 0 modules |
| Level 2 Constructs | Multigene constructs from multiple Level 1 modules |
| Assembly Method | One-pot Golden Gate cloning with type IIS enzymes |
| Efficiency | High cloning efficiency with 95-100% success rate |
| Applications | Gene stacking, metabolic engineering, construct libraries |
This modular cloning system presents a significant advancement in synthetic biology, providing a robust platform for the design and optimization of genetic constructs.
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Literatures Citing This Work
- Assembly of custom TALE-type DNA binding domains by modular cloning. - Robert Morbitzer;Janett Elsaesser;Jens Hausner;Thomas Lahaye - Nucleic acids research (2011)
- Assembly of designer TAL effectors by Golden Gate cloning. - Ernst Weber;Ramona Gruetzner;Stefan Werner;Carola Engler;Sylvestre Marillonnet - PloS one (2011)
- GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. - Alejandro Sarrion-Perdigones;Erica Elvira Falconi;Sara I Zandalinas;Paloma Juárez;Asun Fernández-del-Carmen;Antonio Granell;Diego Orzaez - PloS one (2011)
- Reiterative Recombination for the in vivo assembly of libraries of multigene pathways. - Laura M Wingler;Virginia W Cornish - Proceedings of the National Academy of Sciences of the United States of America (2011)
- A transcription activator-like effector toolbox for genome engineering. - Neville E Sanjana;Le Cong;Yang Zhou;Margaret M Cunniff;Guoping Feng;Feng Zhang - Nature protocols (2012)
- Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination. - Lin Zhang;Guoping Zhao;Xiaoming Ding - Scientific reports (2011)
- High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants. - Pu Yan;Wentao Shen;XinZheng Gao;Xiaoying Li;Peng Zhou;Jun Duan - PloS one (2012)
- A rapid cloning method employing orthogonal end protection. - Arjen J Jakobi;Eric G Huizinga - PloS one (2012)
- The PLOS ONE synthetic biology collection: six years and counting. - Jean Peccoud;Mark Isalan - PloS one (2012)
- Precision editing of large animal genomes. - Wenfang Spring Tan;Daniel F Carlson;Mark W Walton;Scott C Fahrenkrug;Perry B Hackett - Advances in genetics (2012)
... (578 more literatures)
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