TRINS: a method for gene modification by randomized tandem repeat insertions.

Area of Science:

• Protein Engineering
• Molecular Biology
• Synthetic Biology

Background:

• Protein evolution relies on both point mutations and backbone modifications (insertions/deletions).
• Current laboratory methods primarily focus on point mutations, limiting exploration of natural protein diversification strategies.
• Short insertions-by-duplication are common in nature but difficult to generate in vitro.

Purpose of the Study:

• To develop a novel laboratory method for generating gene libraries with insertions-by-duplication.
• To enable the creation of diverse protein variants by mimicking natural evolutionary processes.
• To explore sequence space inaccessible through existing protein diversification techniques.

Main Methods:

• Introduced a new procedure named Tandem Repeat Insertion (TRINS).
• TRINS utilizes rolling-circle amplification to generate tandem repeats of random gene fragments.
• These repeats are concurrently incorporated into the target gene at random positions and variable lengths (3-150 bp).

Main Results:

• Successfully generated gene libraries with insertions-by-duplication.
• Demonstrated the method's efficacy by constructing three distinct gene libraries.
• Selected insertion variants of TEM-1 β-lactamase, showcasing the method's applicability.

Conclusions:

• TRINS provides a powerful new tool for protein diversification, enabling the generation of libraries with unique insertion patterns.
• This method expands the accessible sequence space for protein engineering, facilitating the discovery of novel protein functions.
• TRINS effectively complements existing methods by enabling the introduction of backbone modifications previously challenging to achieve in vitro.