Towards the construction of high-quality mutagenesis libraries
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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.
KeywordsDirected 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.
The raw data of the fluorescence of the transformations in Table 1.
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