Helper Plasmid for Stable Yeast Genome Transformation
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Solution Overview
Problem
Existing methods for introducing a gene of interest into yeast are either highly efficient but unstable, or stable but inefficient, and require complex steps involving nucleases and guide RNA.
Innovation Solution
A method using a helper plasmid for transformation that incorporates linear genome-introduced nucleic acid fragments with a gene of interest, paired homologous recombination sequences, and endonuclease target sequences, allowing for efficient and stable incorporation of the gene into the yeast genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a circular plasmid is used to introduce a gene of interest into yeast, then transformation efficiency is high (approximately 10^-2), but the plasmid may be detached and stable recombinant yeast cannot be produced
Solution Approach 1:
The plasmid is divided into two separate components: a circular helper plasmid that remains in the yeast cell and provides endonuclease activity, and a linear DNA fragment containing the gene of interest with homologous recombination sequences. This segmentation allows the helper plasmid to facilitate stable integration without itself being integrated, resolving the contradiction between transformation efficiency and stability.
Solution Approach 2:
The helper plasmid acts as an intermediary that expresses endonuclease to cleave specific DNA sequences, thereby facilitating the integration of the linear gene fragment into the yeast genome. This intermediary mechanism enables stable integration without requiring direct plasmid integration, maintaining both efficiency and stability.
2Reliability
If a linear vector is used to introduce a gene of interest into yeast, then stable incorporation into the genome is achieved, but transformation efficiency is low (approximately 10^-6)
Solution Approach 1:
The target sequence for endonuclease cleavage is预先 introduced into the linear DNA fragment along with homologous recombination sequences. This preliminary preparation of the DNA structure with built-in cleavage sites and recombination sequences enables efficient and stable integration without requiring complex subsequent steps.
Solution Approach 2:
The helper plasmid expressing endonuclease serves as an intermediary that catalyzes the cleavage and integration process. This intermediary enzyme system dramatically enhances the transformation efficiency of linear DNA fragments while maintaining stable genomic integration.
3Productivity
If endonuclease target sequences are introduced into the genome and double strands are cleaved to improve gene introduction efficiency, then transformation efficiency improves (approximately 10^2 to 10^-1), but the process becomes complicated requiring various steps in addition to DNA fragment production and transformation
Solution Approach 1:
The helper plasmid combines multiple functions into a single construct: it contains the endonuclease gene, the target sequence for cleavage, and elements for plasmid maintenance. This merging eliminates the need for separate steps of target sequence introduction and nuclease application, simplifying the overall process while maintaining high efficiency.
Solution Approach 2:
The helper plasmid is designed to be universally applicable for introducing different genes of interest. Once established in the yeast cell, it can facilitate the integration of various linear DNA fragments containing different genes, reducing the need for repeated complex procedures for each transformation experiment.
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 method significantly improves the efficiency of gene incorporation into the yeast genome, achieving transformation efficiencies approximately 50 to 240 times higher than without the helper plasmid, while ensuring stability of the recombinant yeast.
Implementation Method 1
a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragments
Implementation Method 2
a pair of endonuclease target sequences outside of the pair of 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 method for producing a transformant, comprising a step of introducing into a host, linear genome-introduced nucleic acid fragment(s) comprising a gene of interest and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragment(s), and then selecting a transformant, in which the gene of interest is incorporated into the predetermined position in the host genome and the gene of interest is expressed therein.