Helper Plasmid Counter Selection for Stable Yeast Transformation
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Solution Overview
Problem
Current methods for introducing a gene of interest into a yeast host are either inefficient or unstable, with circular plasmids being easily detached and linear vectors requiring complex steps for homologous recombination, making it difficult to produce a stable transformant efficiently.
Innovation Solution
A method involving a helper plasmid with homologous recombination sequences and a counter selection marker is used to incorporate a linear nucleic acid fragment with a gene of interest into the host genome, where the counter selection marker induces death in cells where the gene is not properly integrated, ensuring efficient and stable transformation.
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 high (approximately 10^-2), but the plasmid may be detached and stable recombinant yeast cannot be produced
Solution Approach 1:
The system is divided into two separate components: a circular helper plasmid that provides high transformation efficiency and a linear nucleic acid fragment containing the gene of interest that ensures stable genomic integration. The helper plasmid and linear fragment work together, with the linear fragment being incorporated into the host genome while the helper plasmid can be detached, thus resolving the contradiction between transformation efficiency and stability.
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) and complex steps are required
Solution Approach 1:
A circular helper plasmid acts as an intermediary carrier that facilitates the introduction of the linear nucleic acid fragment containing the gene of interest into the yeast host. The helper plasmid enables efficient transformation while the linear fragment ensures stable genomic integration, thus resolving the contradiction between transformation efficiency and stability.
3Productivity
If endonuclease target sequences are introduced into the genome to improve homologous recombination efficiency, then transformation efficiency increases (approximately 10^-2 to 10^-1), but the process becomes complicated and requires various additional steps
Solution Approach 1:
The endonuclease target sequences and endonuclease expression system are extracted from the linear nucleic acid fragment and placed in a separate circular helper plasmid. This allows the linear fragment to focus on providing the gene of interest and homologous recombination sequences for stable integration, while the helper plasmid handles the efficiency-enhancing endonuclease functions, thus reducing overall process complexity.
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 allows for the efficient production of stable transformants by ensuring the gene of interest is correctly integrated into the host genome, reducing false positives and increasing transformation efficiency.
Implementation Method 1
a counter selection marker, wherein the counter selection marker functions to induce the death of a host cell comprising the helper plasmid for transformation into which the linear nucleic acid fragment is incorporated
Implementation Method 2
a pair of homologous recombination sequences to incorporate the linear nucleic acid fragment... a pair of homologous recombination sequences that undergoes homologous recombination between a region outside of the gene of interest and a given site of the genome
Data Source
AI summary
This invention is intended to simply and efficiently produce a stable transformant comprising a gene of interest incorporated into the genome. By introducing linear nucleic acid fragments for gemone-introduction comprising a gene of interest and a helper plasmid for transformation comprising a pair of homologous recombination sequences to incorporate the fragment and functioning a counter selection marker, a host comprising the helper plasmid for transformation into which the linear nucleic acid fragment is incorporated. Results in death.