Biotechnology Letters

, Volume 40, Issue 7, pp 1101–1107 | Cite as

Towards the construction of high-quality mutagenesis libraries

  • Heng Li
  • Jing Li
  • Ruinan Jin
  • Wei Chen
  • Chaoning Liang
  • Jieyuan Wu
  • Jian-Ming Jin
  • Shuang-Yan TangEmail author
Original Research Paper



To improve the quality of mutagenesis libraries in directed evolution strategy.


In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells.


This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.


Directed evolution Multiple-plasmid transformant Mutagenesis library Transformation Transformation efficiency 



This work was supported by the National Natural Science Foundation of China (Grant Nos. 21472234, 31501037, 31160017 and 21506245), and the CAS/SAFEA International Partnership Program for Creative Research Teams. We thank Dr. Guoxia Liu for her contributions in this work.

Supporting Information

The raw data of the fluorescence of the transformations in Table 1.

Supplementary material

10529_2018_2559_MOESM1_ESM.xlsx (32 kb)
Supplementary material 1 (XLSX 32 kb)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Heng Li
    • 1
    • 3
  • Jing Li
    • 1
    • 3
  • Ruinan Jin
    • 1
  • Wei Chen
    • 1
  • Chaoning Liang
    • 1
  • Jieyuan Wu
    • 1
    • 3
  • Jian-Ming Jin
    • 2
  • Shuang-Yan Tang
    • 1
    Email author
  1. 1.CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, State Key Laboratory of Microbial ResourcesChinese Academy of SciencesBeijingChina
  2. 2.Beijing Key Laboratory of Plant Resources Research and DevelopmentBeijing Technology and Business UniversityBeijingChina
  3. 3.University of Chinese Academy of SciencesBeijingChina

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