EPSPS Mutant Gene Screening via E. coli Intermediary Host
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Solution Overview
Problem
Current methods for screening glyphosate-resistant mutant genes from plants are time-consuming and require large acreage, leading to inefficiencies and unreliable results due to potential resistance development from host strain mutations.
Innovation Solution
A method involving the construction of EPSPS gene-and-C-P lyase genes-deleted E. coli strains, introduction of exogenous EPSPS genes from target plants, and mutagenesis to create mutant libraries, followed by screening on glyphosate-containing media to identify resistant strains, ensuring resistance originates from exogenous genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional plant mutagenesis and screening methods are used, then glyphosate-resistant mutant genes can be obtained, but the process takes a long time and requires large acreage
Solution Approach 1:
The patent uses E. coli as an intermediary host organism to express and screen plant EPSPS genes. The E. coli strain is engineered to lack endogenous EPSPS activity, making it dependent on the introduced plant EPSPS gene for survival. This intermediary system allows rapid bacterial growth and screening while ensuring that any observed glyphosate resistance must originate from the introduced plant gene, not host mutations.
Solution Approach 2:
The patent changes the biological system from whole plants to bacterial cells, fundamentally altering growth parameters. Bacteria grow much faster than plants and can be cultured in small volumes, transforming a process that previously required large acreages and long growing seasons into one that can be completed in days using minimal space.
2Reliability
If traditional plant mutagenesis and screening methods are used, then glyphosate-resistant mutant genes can be obtained, but large acreage is required
Solution Approach 1:
The patent uses E. coli as an intermediary host organism to express and screen plant EPSPS genes. The E. coli strain is engineered to lack endogenous EPSPS activity, making it dependent on the introduced plant EPSPS gene for survival. This intermediary system allows rapid bacterial growth and screening while ensuring that any observed glyphosate resistance must originate from the introduced plant gene, not host mutations.
Solution Approach 2:
The patent changes the biological system from whole plants to bacterial cells, fundamentally altering growth parameters. Bacteria grow much faster than plants and can be cultured in small volumes, transforming a process that previously required large acreages and long growing seasons into one that can be completed in days using minimal space.
3Reliability
If CP4 gene is used for glyphosate resistance, then high resistance is achieved, but the crop is considered transgenic and faces public acceptance issues
Solution Approach 1:
The patent focuses on creating glyphosate resistance within the plant's own EPSPS gene through site-directed mutagenesis, rather than introducing foreign genes. This localized modification approach allows the resistance trait to be embedded in the plant's native genome, producing non-transgenic crops that maintain high glyphosate resistance while avoiding public acceptance issues associated with transgenic organisms.
Data Source
AI summary
Provided are a glyphosate-resistant gene screening method, an EPSPS mutant gene having glyphosate resistance screened by the method, an EPSPS and C-P Lyase deficient strain and a use thereof.