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Available methods for assembling expression cassettes for synthetic biology

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Abstract

Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.

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Acknowledgements

Grants from the National Science Foundation of China (31000054, 30873190) and the Fundamental Research Funds for the Central Universities (No. JUSRP10917) supported this research.

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China

    Tianwen Wang, Guocheng Du & Jian Chen

  2. School of Biotechnology, and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 214122, China

    Xingyuan Ma & Aitao Li

  3. Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, 266555, China

    Hu Zhu

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  1. Tianwen Wang
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Correspondence to Xingyuan Ma.

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Wang, T., Ma, X., Zhu, H. et al. Available methods for assembling expression cassettes for synthetic biology. Appl Microbiol Biotechnol 93, 1853–1863 (2012). https://doi.org/10.1007/s00253-012-3920-8

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