Targeted Mutagenesis via Error-Polmerase and Nickase Plasmid System
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Solution Overview
Problem
Current methods for targeted mutagenesis in bacteria are cumbersome and slow, particularly when attempting to mutate specific genes for degrading recalcitrant compounds like polyaromatic hydrocarbons and chlorinated solvents, as they rely on non-specific mutation techniques.
Innovation Solution
The method involves expressing an error-prone DNA polymerase in a controlled manner in a host bacterial strain, in conjunction with a nickase, and placing a targeted nucleic acid on an episome or plasmid with nickase recognition sequences, allowing for selective mutation of specific genes at significantly higher rates than chromosomal genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If non-specific mutation techniques are used, then the mutagenesis process is simple, but the mutation rate for specific genes is low and the process is slow
Solution Approach 1:
The patent segments the bacterial genome by placing the target gene on a separate plasmid, physically separating it from the chromosomal DNA. This allows the error-prone polymerase to selectively mutate only the plasmid-borne target gene while leaving the chromosomal genes unchanged, thereby increasing the mutation rate for specific genes without requiring complex mutagenesis systems.
Solution Approach 2:
The patent applies local quality by creating a localized high-mutation-rate zone on the plasmid while maintaining normal mutation rates elsewhere in the genome. The error-prone polymerase is selectively expressed to mutate only the target gene region on the plasmid, concentrating mutational activity where needed without affecting the rest of the bacterial genome.
2Productivity
If error-prone polymerase is over-expressed, then mutation rate increases, but cell viability decreases at very high levels
Solution Approach 1:
The patent employs dynamic control of error-prone polymerase expression through an inducible promoter system. The polymerase is normally repressed but can be induced to high levels only when needed for mutagenesis. This dynamic regulation allows the system to achieve high mutation rates during induction while maintaining cell viability during normal growth, resolving the contradiction between productivity and reliability.
Solution Approach 2:
The patent implements periodic action by cycling the expression of the error-prone polymerase between repressed and induced states. The inducible promoter allows temporary activation of high mutation rates followed by return to normal expression levels, enabling repeated mutagenesis cycles without permanently compromising cell viability.
3Manufacturing precision
If targeted mutagenesis is achieved through plasmid-based methods, then specific gene mutation is enhanced, but the method becomes more complex
Solution Approach 1:
The patent applies universality by using a plasmid-based system that can accommodate any target gene of interest. The same plasmid construction strategy and error-prone polymerase expression system can be used to mutate different genes by simply changing the insert, making the method universally applicable without requiring gene-specific complex procedures for each target.
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 enables rapid and targeted mutagenesis, increasing mutation rates within specific genes, facilitating the development of bacterial strains that can adapt quickly to stressors and efficiently degrade environmental contaminants or convert low-value feedstocks into valuable products.
Implementation Method 1
expressing an error-prone polymerase in a controlled manner in a host bacterial strain... allowing for selective mutation of specific genes
Implementation Method 2
in conjunction with a nickase, and placing a targeted nucleic acid on an episome or plasmid which contains one or more nickase recognition sequences
Data Source
AI summary
This disclosure provides methods and compositions for targeted mutagenesis of specific genes in a bacterial strain. By inducibly over-expressing error-prone polymerases such as Pol IV or Pol V in conjunction with nickase in a bacterial strain, and housing the targeted gene(s) on an episome or plasmid which contains one or more nickase recognition sequences, the targeted gene(s) can be selectively mutated at rates significantly greater than genes contained on the chromosome. The methods disclosed herein are useful for engineering desirable bacterial phenotypes and novel strains, including for example strains useful for treating or degrading waste and/or environmental contaminants, for optimizing bioprocesses, and for converting low-value feed-stock into value-added products.