Orthogonal DNA Replication for Targeted Continuous Evolution
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Solution Overview
Problem
Current laboratory evolution methods face constraints such as slow organismal evolution and untargeted mutations, as natural evolution is slow and random, and ex vivo mutagenesis is labor-intensive and not capable of continuous or massive parallelization.
Innovation Solution
Development of orthogonal nucleic acid replication systems using mutant polymerases that can continuously mutate user-defined genes in vivo, with altered mutation rates and specificity to plasmids, allowing for independent replication of multiple plasmids without affecting genomic nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ex vivo mutagenesis is used to control mutation rate and target specific genes, then manufacturing precision and adaptability are improved, but productivity and ease of operation deteriorate due to labor-intensive DNA extraction, mutation, and transformation steps for each round of evolution
Solution Approach 1:
The invention divides the evolution process into separate orthogonal replication systems, each with its own mutant polymerase that operates independently on specific plasmids. This segmentation allows simultaneous parallel evolution of multiple genes without cross-interference, resolving the contradiction between precise mutation control and evolution speed
Solution Approach 2:
The orthogonal replication systems enable continuous in vivo evolution without requiring labor-intensive ex vivo intervention steps. The mutant polymerases continuously replicate plasmids and introduce mutations throughout cell division, maintaining useful evolutionary action across generations without stopping for DNA extraction and transformation
2Ease of operation
If natural evolution is allowed to occur in vivo, then ease of operation is improved, but manufacturing precision and adaptability worsen due to slow evolution and random untargeted mutations across the entire genome
Solution Approach 1:
Each orthogonal replication system is designed with local quality - a mutant polymerase that acts specifically on its assigned plasmid with a defined mutation rate. This localized control ensures mutations occur only in the intended target gene on the specific plasmid, not randomly across the entire genome, while maintaining in vivo simplicity
Solution Approach 2:
The mutant polymerases serve as intermediaries that bridge the gap between simple in vivo evolution and precise targeted mutagenesis. These engineered enzymes mediate the replication process, introducing mutations at controlled rates specifically on plasmids while leaving genomic DNA untouched, thus achieving both ease of operation and manufacturing precision
3Productivity
If mutant polymerases with high mutation rates are used to accelerate evolution, then productivity is improved, but reliability worsens because such high mutation rates would result in host cell death if applied to genomic nucleic acids
Solution Approach 1:
The invention extracts the mutagenic function from the genomic replication system and places it in separate orthogonal plasmid replication systems. The mutant polymerases are taken out of the context of genomic DNA replication and applied only to plasmids, allowing high mutation rates to be used productively without threatening host cell viability through genomic damage
Solution Approach 2:
The replication system is segmented into multiple independent orthogonal systems, each with its own mutant polymerase operating on specific plasmids. This segmentation isolates the high-risk mutagenic activity to non-essential plasmid DNA, protecting essential genomic DNA from excessive mutations while maintaining high evolutionary productivity in the plasmid-borne genes
Data Source
AI summary
The invention provides compositions comprising highly error-prone polymerases and methods of using the polymerases for rapid evolution of a nucleic acid sequence within host cells. The invention further provides a versatile synthetic biology platform for manipulating DNA replication inside a cell. The invention also provides a mutually orthogonal replication system for manipulating and tuning DNA replication of multiple nucleic acid molecules using the error-prone polymerases.