A Golden Gate compatible system for continuous directed evolution in E. coli.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Directed evolution is a powerful protein engineering method that explores gene variants for improved functions.
• Continuous directed evolution enhances this process by linking hypermutation to selection within single cells.
• Existing methods can be inflexible and labor-intensive, limiting their widespread application.

Purpose of the Study:

• To develop a versatile and accessible modular toolkit for continuous directed evolution.
• To adapt viral RNA polymerases for gene-specific hypermutation and functional selection.
• To enable researchers to construct custom plasmids for diverse evolutionary campaigns.

Main Methods:

• Utilized Golden Gate assembly for modular plasmid construction.
• Developed a toolkit featuring deaminase-fused viral RNA polymerase.
• Incorporated an alternative RNA polymerase from phage SP6 to demonstrate versatility.

Main Results:

• The toolkit facilitates continuous directed evolution with enhanced accessibility and versatility.
• Demonstrated gene-specific mutation introduction using the engineered RNA polymerase.
• Successfully built custom plasmids for complex evolutionary strategies.

Conclusions:

• The presented toolkit significantly improves the ease and flexibility of continuous directed evolution.
• This advancement expands the synthetic biology toolbox for protein engineering.
• Enables broader research into discovering novel gene functions and optimizing protein performance.