Transformation Plasmid with Endonuclease Sites for Stable Genomic Integration
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Solution Overview
Problem
Current methods for introducing a gene of interest into a host genome, such as yeast or Escherichia coli, are inefficient and complex, often requiring additional steps like nuclease introduction or guide RNA production, and do not reliably produce stable transformants.
Innovation Solution
A plasmid for transformation is designed with homologous recombination sequences and endonuclease target sequences, along with a target-specific endonuclease gene that cleaves the endonuclease target sequences, allowing for efficient incorporation of the gene of interest into the host genome via homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a circular plasmid is used to introduce a gene of interest into a yeast host, then transformation efficiency is improved (approximately 10^-2), but the plasmid may detach and stable recombinant yeast cannot be produced
Solution Approach 1:
The plasmid is segmented into specific functional modules: homologous recombination sequences (LR sequences) that enable integration into the host genome, endonuclease target sequences (DdeI sites) for site-specific cleavage, and a gene of interest. This segmentation allows the plasmid to efficiently transform yeast while ensuring stable genomic integration through the LR sequences.
Solution Approach 2:
The plasmid is pre-configured with endonuclease target sequences (DdeI sites) at specific locations before transformation. The endonuclease is then expressed to cleave the plasmid DNA at these predetermined sites, creating double-strand breaks that facilitate homologous recombination and stable genomic integration of the gene of interest.
2Reliability
If a linear vector is used to introduce a gene of interest into a yeast host, then stable incorporation into the genome is achieved, but transformation efficiency is low (approximately 10^-6)
Solution Approach 1:
The plasmid is pre-configured with endonuclease target sequences (DdeI sites) flanking the homologous recombination sequences. Expression of the endonuclease creates double-strand breaks at these predetermined sites, converting the plasmid into a linear structure in situ within the yeast cell, thereby enabling efficient homologous recombination without requiring external linearization.
Solution Approach 2:
The endonuclease acts as an intermediary that converts the circular plasmid into a linear structure by cleaving at specific DdeI target sequences. This intermediary action enables the plasmid to achieve both efficient transformation (through controlled linearization) and stable genomic integration (through homologous recombination mediated by the LR sequences).
3Productivity
If target-specific endonuclease or CRISPR-Cas9 is used to improve homologous recombination efficiency, then efficiency is improved (approximately 10^-2 to 10^-1), but the process becomes complicated requiring additional steps
Solution Approach 1:
The invention merges multiple functions into a single plasmid construct: the homologous recombination sequences (LR sequences) for genomic integration, the endonuclease target sequences (DdeI sites) for controlled cleavage, and the gene of interest. This unified plasmid design eliminates the need for separate nuclease expression vectors and guide RNA construction, simplifying the overall process while maintaining high efficiency.
Solution Approach 2:
The plasmid construct serves multiple functions simultaneously: it acts as a transformation vector, contains the gene of interest, provides homologous recombination sequences for integration, and includes endonuclease target sequences for site-specific cleavage. This multi-functionality eliminates the need for multiple separate components required by CRISPR-Cas9 or TALEN systems.
4Productivity
If Red recombinase operon is introduced into E. coli to improve homologous recombination efficiency, then efficiency is improved, but the work becomes complicated requiring transformation twice
Solution Approach 1:
The endonuclease target sequences (DdeI sites) are pre-inserted into the plasmid construct before transformation. Upon expression of the endonuclease in the yeast host, these predetermined sites are cleaved to generate double-strand breaks that immediately facilitate homologous recombination, eliminating the need for a second transformation step required by Red recombinase systems.
Solution Approach 2:
The invention extracts and utilizes the essential function of site-specific cleavage through endonuclease recognition sequences directly within the transformation plasmid. This eliminates the need for separate recombination enzyme systems (如Red recombinase) and multiple transformation steps, achieving efficient genomic integration in a single transformation event.
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 simplifies and enhances the production of stable transformants by efficiently incorporating the gene of interest into the host genome, improving transformation efficiency and reducing the complexity of the process.
Implementation Method 1
a target-specific endonuclease gene that specifically cleaves the double strands of the endonuclease target sequences
Implementation Method 2
a pair of homologous recombination sequences sandwiching the site... the gene of interest is incorporated into the genome via the homologous recombination sequences
Data Source
AI summary
A stable transformant, in which a gene of interest is incorporated into the genome, is simply and efficiently produced. A plasmid for transformation comprises a site into which a gene of interest is to be incorporated, a pair of homologous recombination sequences sandwiching the site, and a pair of endonuclease target sequences sandwiching the pair of homologous recombination sequences.