SYSTEM FOR THE DIRECTED EVOLUTION OF BIOMOLECULES IN MULTI-BODY COMPLEXES

Smart overview of the Invention

A novel system and method have been developed for the continuous evolution of biomolecules that form multi-body complexes, specifically those involving three or four molecules. This approach aims to enhance the efficiency of evolving protein functions, which has traditionally been limited to proteins with enzymatic roles. The methodology leverages recent advancements in bacterial growth maintenance and low-volume culture systems to facilitate the evolution process.

Background and Challenges

Continuous evolution has proven to be a rapid technique for improving protein functionality, yet it has primarily focused on enzymatic proteins. Previous methods faced limitations due to challenges in optimizing biological activity linked to gene expression, particularly in complex binding interactions. These challenges included the need for precise control of small molecule inducers and the inability to create new evolution circuits effectively.

Methodology for Evolving Multi-Body Complexes

The method involves preparing an evolution circuit with multiple molecules that can form a multi-body complex. Key steps include:

• Forming the multi-body complex within the circuit.
• Determining equilibrium conditions for each molecule to achieve an equimolar ratio.
• Selecting and maintaining evolution conditions based on these equilibrium states.
• Evolving the molecules under the maintained conditions.

Automation and Optimization Techniques

The process may utilize automated liquid-handling robots for high-throughput preparation of evolution circuits. The method also includes culturing cells capable of protein evolution, maintaining cell density through feedback controls, and inducing production of necessary molecules. Furthermore, it explores various combinations of inducing agents to achieve kinetic equilibrium among the molecules involved in the multi-body complex.

Potential Applications and Innovations

This system opens up new avenues for evolving proteins that participate in complex interactions, such as protein-protein or protein-small molecule binding. It allows for the incorporation of detectable reporters to monitor progress and optimize conditions further. By enabling the evolution of more intricate biomolecular structures, this methodology could significantly impact fields such as biotechnology, pharmaceuticals, and synthetic biology.