Low Copy Cosmid Vector for Plant Transformation
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Solution Overview
Problem
Current cosmid vectors face challenges in efficiently cloning and transferring large DNA fragments into plants, particularly due to high copy number issues in E.coli and Agrobacterium, leading to low transformation efficiency and complex cloning operations, which hampers map-based cloning and gene transfer processes.
Innovation Solution
Development of novel cosmid vectors with a full length of 15 kb or less, containing an IncP plasmid origin of replication, trfA1 gene, oriT, incC1 gene, a cos site outside the T-DNA, and a selectable marker, optimized for stable maintenance and efficient transfer in E.coli and Agrobacterium, allowing for the cloning of DNA fragments up to 25-40 kb.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If cosmid vectors with IncP plasmid origin of replication are used, then stable maintenance in E.coli and Agrobacterium is achieved, but high copy number leads to low transformation efficiency
Solution Approach 1:
The patent changes the copy number parameter of the plasmid by using IncP plasmid origin of replication, which naturally provides low copy number (1-2 copies per cell) compared to other origins like ColE1. This parameter change simultaneously achieves stable maintenance in both E.coli and Agrobacterium while improving transformation efficiency, as the low copy number reduces metabolic burden and increases transformation reliability
2Adaptability or versatility
If vectors with size of 24-30 kb are used, then plant transformation elements are included, but the size of clonable DNA fragments is restricted to about 13-22 kb
Solution Approach 1:
The patent segments the vector into modular components: IncP plasmid backbone (providing replication and stability), T-DNA borders (providing plant transformation capability), and a large insert region. This segmentation allows the vector to accommodate unusually large DNA fragments (25-40 kb) while maintaining all necessary plant transformation elements, effectively resolving the size limitation of conventional vectors
3Ease of manufacture
If lambda phage packaging reaction is used for cloning, then easy cloning of large genomic fragments is achieved, but the total size of vector and insert is restricted to 40-50 kb
Solution Approach 1:
The patent creates a universal cosmid vector that combines multiple functions: it maintains the cos site for lambda phage packaging reaction (enabling easy cloning), includes T-DNA borders for plant transformation, and uses IncP plasmid origin for stable maintenance in both E.coli and Agrobacterium. This multi-functionality allows the vector to accommodate larger inserts (25-40 kb) while preserving the ease of cloning through packaging reaction
Data Source
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AI summary
The present invention aims to provide novel vectors for plant transformation. The vectors of the present invention are cosmid vectors having a full length of 15 kb or less characterized in that: 1) they contain an origin of replication of an IncP plasmid, but do not contain any origin of replication of other plasmid groups; 2) they contain the trfA1 gene of an IncP plasmid; 3) they contain an oriT of an IncP plasmid; 4) they contain the incC1 gene of an IncP plasmid; 5) they contain a cos site of lambda phage and the cos site is located outside the T-DNA; 6) they contain a drug resistance gene expressed in E.coli and a bacterium of the genus Agrobacterium; 7) they contain a T-DNA right border sequence of a bacterium of the genus Agrobacterium; 8) they contain a T-DNA left border sequence of a bacterium of the genus Agrobacterium; 9) they contain a selectable marker gene for plant transformation located between 7) and 8) and expressed in a plant; and 10) they contain restriction endonuclease recognition site(s) located between 7) and 8) for cloning a foreign gene.