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Methods Mol Biol. 2018;1772:205-212. doi: 10.1007/978-1-4939-7795-6_11.

Combinatorial Evolution of DNA with RECODE.

Kang Z1,2,3, Ding W4, Jin P4, Du G4,5, Chen J4,5.

Author information

1
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China. zkang@jiangnan.edu.cn.
2
Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, China. zkang@jiangnan.edu.cn.
3
The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China. zkang@jiangnan.edu.cn.
4
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
5
Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, China.

Abstract

In past decades, DNA engineering protocols have led to the rapid development of synthetic biology. To engineer the natural proteins, many directed evolution methods based on molecular biology have been presented for generating genetic diversity or obtaining specific properties. Here, we provide a simple (PCR operation), efficient (larger amount of products), and powerful (multiple point mutations, deletions, insertions, and combinatorial multipoint mutagenesis) RECODE method, which is capable of reediting the target DNA flexibly to restructure regulatory regions and remodel enzymes by using the combined function of the thermostable DNA polymerase and DNA ligase in one pot. RECODE is expected to be an applicable choice to create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways.

KEYWORDS:

Combinatorial multiple mutagenesis; Directed evolution; Pathway optimization; Single-stranded DNA oligonucleotides; Synthetic biology

PMID:
29754230
DOI:
10.1007/978-1-4939-7795-6_11
[Indexed for MEDLINE]

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