Objectives: To improve the quality of mutagenesis libraries in directed evolution strategy.
Results: 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.
Conclusions: 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.
Keywords: Directed evolution; Multiple-plasmid transformant; Mutagenesis library; Transformation; Transformation efficiency.