Single-Plasmid Construction of Two-Component Plant Viral Vectors
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Solution Overview
Problem
Current methods for constructing two-component plant viral vectors, such as TRV and CLCrV, require separate plasmids and involve complex processes like Agrobacterium transformation, increasing workload and contamination risk, especially in high-throughput experiments.
Innovation Solution
Concatenating two-component virus genomes into a single plasmid system using adapter sequences, simplifying the construction process and reducing contamination risks through a single PCR product.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If two-component viral vectors are delivered using separate plasmids through Agrobacterium transformation, then the viral vectors can be constructed and delivered to plant cells, but the experimental workload increases and contamination risk rises
Solution Approach 1:
The patent merges two separate plasmid delivery steps into a single plasmid delivery step by combining the genomes of two-component viral vectors (TRV or CLCrV) into one plasmid. This single plasmid contains all necessary viral components and can be transformed into Agrobacterium and delivered to plant cells in one transformation step, eliminating the need for separate transformations and mixing steps, thereby reducing contamination risk and experimental workload
2Ease of manufacture
If two-component viral vectors are constructed with separate plasmids, then the viral components can be properly assembled, but the construction process becomes complex and time-consuming
Solution Approach 1:
The patent combines the construction of two separate plasmids into a single plasmid construction process. The single plasmid is designed to contain both viral genome components with appropriate promoters and regulatory elements, allowing simultaneous expression of all viral components after transformation, thereby simplifying the construction process and reducing the time required
3Reliability
If multiple Agrobacterium strains are used for delivering two-component viral vectors, then the complete viral system can be reconstituted, but the risk of cross-contamination between bacterial solutions increases
Solution Approach 1:
The patent merges the delivery function of multiple Agrobacterium strains into a single Agrobacterium strain. The single plasmid contains all necessary viral components that were previously distributed across multiple plasmids in different strains, allowing one Agrobacterium strain to deliver the complete viral system, thereby eliminating cross-contamination risks associated with mixing multiple bacterial solutions
Data Source
AI summary
A method for simply constructing a two-component virus vector and related applications. The two-component plant viral vector is optimized, so that a two-component virus genome is placed in a single plasmid. Due to the use of the single plasmid, the activation, resuspension and mixing processes of auxiliary bacteria are avoided, the workload of an Agrobacterium experimental stage is simplified by half, the potential cross-contamination risk in the bacteria mixing process is avoided, and the virus infectivity is improved, so that the use of the two-component viral vector is greatly simplified.