Continuous Directed Evolution Using Phage-Driven Selection
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Solution Overview
Problem
Conventional directed evolution methods are labor-intensive and time-consuming, limiting the number of rounds of mutation, selection, and replication, making them impractical for complex evolution processes in the laboratory.
Innovation Solution
A platform for continuous directed evolution that enables the transfer of genes between host cells through a modified viral life cycle, such as phage-assisted continuous evolution (PACE), allowing multiple cycles of replication, mutation, and selection without human intervention, using systems like phage vectors that depend on the activity of the gene of interest to drive expression and transfer.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional discrete directed evolution methods are used, then mutation, selection, and replication can be performed with researcher control, but the process becomes labor-intensive and time-consuming, limiting the number of rounds to a modest number
Solution Approach 1:
The patent implements continuous directed evolution by establishing an in vitro evolution system where ribozymes continuously replicate and evolve without discrete researcher intervention. The system maintains continuous mutation, selection, and replication cycles through automated flow-through reactors, eliminating the labor-intensive discrete cycles of conventional methods and enabling thousands of evolution rounds to occur simultaneously with minimal researcher time or effort
Solution Approach 2:
The evolution system is designed to be self-sustaining, where the ribozymes themselves drive their own evolution through autonomous replication and selection processes. The system automatically performs mutation generation, selection of functional variants, and amplification without requiring researcher intervention at each step, allowing the evolution process to serve itself and dramatically increasing the number of rounds achievable
2Productivity
If continuous directed evolution is implemented to enable enormous numbers of rounds, then productivity increases dramatically, but the system complexity and difficulty of adaptation to different biomolecules increases
Solution Approach 1:
The patent develops a universal continuous evolution platform that can be applied to evolve different types of biomolecules including ribozymes, proteins, and other catalytic molecules. The system uses generalizable components such as flow-through reactors, automated selection mechanisms, and standardized replication systems that can be adapted to various molecular targets, reducing the complexity burden when applying continuous evolution to different biomolecule classes
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach accelerates the evolution of biomolecules by enabling dozens of cycles in a single day, achieving evolved proteins with desired activities comparable to or exceeding wild-type performance, such as modified T7 RNA polymerase, by leveraging the phage life cycle's efficiency and selective pressure.
Implementation Method 1
a phage genome, comprising a gene of interest, which replicates and mutates in a flow of host cells
Implementation Method 2
a gene encoding a phage protein required for the production of infectious phage particles under the control of a conditional promoter the activity of which depends on a gene product encoded by the gene of interest
Implementation Method 3
continuous mutagenesis of the gene(s) of interest
Implementation Method 4
continuous selective replication of genes encoding molecules with a desired activity
Data Source
AI summary
The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.