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Updated: Jun 25, 2025

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
In vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis
Baowei Wang1,2, Yang Liu3,4, Xuelian Bai3,4
1Department of Strategic and Integrative Research, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. wangbw@tib.cas.cn.
Abstract:
Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With the ever-expanding application of directed evolution, different approaches of generating genetic diversity are required to enrich the traditional toolbox. Here we show in vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis (epADS). This approach comprises a three-step process which incorporates base errors randomly generated during chemical synthesis of oligonucleotides under specific conditions into the target DNA. Through this method, 200 ~ 4000 folds of diversification in fluorescent strength have been achieved in genes encoding fluorescent proteins. EpADS has also been successfully used to diversify regulatory genetic parts, synthetic gene circuits and even increase microbial tolerance to carbenicillin in a short time period. EpADS would be an alternative tool for directed evolution which may have useful applications in synthetic biology.
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