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High efficiency and throughput system in directed evolution in vitro of reporter gene

  • Applied Genetics and Molecular Biotechnology
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Abstract

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli β-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50–100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100°C for 30 min.

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Acknowledgements

This research was supported by the Shanghai Rising-Star Program (05QMX1445); Shanghai Natural Science Foundation (04ZR14116); the Shanghai Project for International Scientific and Technological Cooperation (055407068); the Fund of National Natural Science Foundation of China (30471258; 30370987); the Program of Shanghai Subject Chief Scientist (06XD14017); the Shanghai Key Basic Research Project (05DJ14008); the Project of key laboratory of Shanghai (05dz223266; 03dz19311) and the Development Foundation of SAAS (2006-10). We thank Dr. Genlou Sun for a critical review of the manuscript.

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Authors and Affiliations

  1. Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, 2901 Beidi Road, Shanghai, 201106, China

    Ai-Sheng Xiong, Ri-He Peng, Jin-Ge Liu, Jing Zhuang, Fang Xu, Bing Cai & Quan-Hong Yao

  2. College of Bioscience and Biotechnology, Yangzhou University, 88 South Daxue Road, Yangzhou, 225009, China

    Jian-Min Chen

  3. College of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, China

    Ai-Sheng Xiong, Jin-Ge Liu, Jing Zhuang, Yu-Shan Qiao, Fang Xu, Bing Cai & Zhen Zhang

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  1. Ai-Sheng Xiong
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  2. Ri-He Peng
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  3. Jin-Ge Liu
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  4. Jing Zhuang
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  5. Yu-Shan Qiao
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  6. Fang Xu
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  7. Bing Cai
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  8. Zhen Zhang
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  9. Jian-Min Chen
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Correspondence to Jian-Min Chen or Quan-Hong Yao.

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Xiong, AS., Peng, RH., Liu, JG. et al. High efficiency and throughput system in directed evolution in vitro of reporter gene. Appl Microbiol Biotechnol 74, 160–168 (2007). https://doi.org/10.1007/s00253-006-0659-0

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  • DOI: https://doi.org/10.1007/s00253-006-0659-0

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