Yeast Transformant Production Using Recombinase Marker Excision
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Solution Overview
Problem
Existing methods for integrating target genes into yeast hosts are inefficient, particularly when using linear vectors, and require complex verification steps to ensure accurate integration and deletion of nucleic acid fragments.
Innovation Solution
A method utilizing site-specific recombinases and recognition sequences to integrate and delete nucleic acid fragments in yeast hosts, where at least one recognition sequence is provided in a fragment other than the target gene, allowing for simple evaluation of accurate integration based on target gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a linear vector is used to introduce a target gene into yeast host via homologous recombination, then the target gene can be integrated into the genome, but the efficiency of preparing transgenic yeast is very low (approximately 10^-6)
Solution Approach 1:
The nucleic acid fragment is divided into three segments: an upstream region fragment with a 5' homologous recombination sequence, a target gene fragment, and a downstream region fragment with a 3' homologous recombination sequence. This segmentation allows each fragment to be independently designed and optimized for its specific function, improving overall integration efficiency
Solution Approach 2:
Homologous recombination sequences are预先 (pre) provided at the ends of nucleic acid fragments before introduction into the host. These pre-designed homologous regions enable direct integration into the genome without requiring additional processing steps during transformation, thereby increasing efficiency
2Manufacturing precision
If multiple nucleic acid fragments are introduced into the host to integrate a target gene at a particular site, then the target gene can be precisely located, but it becomes complicated to determine whether accurate integration has occurred
Solution Approach 1:
A selection marker gene is introduced as an intermediary element within the nucleic acid fragment structure. This marker gene serves as a verifiable indicator that the entire construct (including upstream and downstream regions) has been correctly integrated into the genome at the intended site
Solution Approach 2:
The selection marker gene provides immediate feedback on integration success through selectable phenotypes. Only cells that have correctly integrated the complete nucleic acid fragment construct (with marker gene) will survive under selective conditions, automatically verifying accurate integration without complex additional assays
3Ease of manufacture
If a selection marker gene is located between homologous recombination sequences to enable removal, then the marker can be removed after integration, but the process becomes more complex
Solution Approach 1:
The selection marker gene removal function is merged with the site-specific recombination system. The same recombination sequences (loxP or FRT) used for initial integration are also used to remove the marker gene, combining two functions (integration and marker removal) into a unified system that uses the same molecular machinery
Solution Approach 2:
The nucleic acid fragment construct is designed to be self-sufficient by including all necessary elements (homologous recombination sequences, target gene, selection marker, and site-specific recombination sequences) in a single integrated structure. This self-contained design enables the fragment to autonomously integrate into the genome and subsequently allow marker removal without requiring additional external components
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
Enables efficient production of transformants by ensuring accurate integration and deletion of nucleic acid fragments, simplifying the verification process and facilitating the marker recycle method.
Implementation Method 1
A site-specific recombinase is an enzyme that has activity of recognizing a particular, short, homologous pair of nucleotide sequences and causing homologous recombination between the pair of nucleotide sequences
Implementation Method 2
a nucleic acid fragment having either one of the pair of homologous recombination sequences corresponding to a particular region of genome DNA, and a nucleic acid fragment having the other of the pair of homologous recombination sequences into a host cell
Data Source
AI summary
The present disclosure concerns evaluation as to whether or not a nucleic acid fragment having a target gene had been accurately integrated into the host genome. A group of nucleic acid fragments comprising a nucleic acid fragment having a target gene is introduced into host cells, and host cells in which the target gene had been cleaved from the genome DNA by the action of a site-specific recombinase are selected.