Crowding Agent-Mediated Genome Transfer via Cell Fusion
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Solution Overview
Problem
Current methods for cloning and manipulating large nucleic acid molecules, such as genomes, are limited by size restrictions and require time-consuming and costly agarose plug procedures, and do not allow for efficient transfer of manipulated genomes back into similar recipient organisms or across different cell types.
Innovation Solution
A method involving direct contact between bacterial donor cells and yeast host cells in the presence of a crowding agent like PEG, allowing for the transfer and modification of intact nucleic acids or genomes greater than 150 kb, including the use of yeast centromeres and autonomously replicating sequences, and the option to modify donor cells to be restriction nuclease or methyltransferase negative for enhanced transfer efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional cloning methods are used, then nucleic acid transfer is achieved, but the size is limited to a few genes and time-consuming agarose plug procedures are required
Solution Approach 1:
The invention extracts and eliminates the agarose plug intermediate step from the conventional cloning process. By using whole bacterial cells as carriers instead of isolating DNA into agarose plugs, the method removes the time-consuming DNA extraction, plug preparation, and embedding steps while directly transferring intact genomes (greater than 150 kb) from donor to recipient cells through cell fusion
Solution Approach 2:
The invention embeds the donor bacterial cell containing the intact genome inside the recipient yeast cell through PEG-induced cell fusion. The donor cell acts as a nested structure that delivers the entire genome directly into the recipient cell, eliminating the need for external DNA isolation and plug preparation steps
2Length of moving object
If conventional cloning methods are used, then nucleic acid transfer is achieved, but costly and time-consuming agarose plug procedures are required
Solution Approach 1:
The invention extracts and eliminates the agarose plug intermediate step from the conventional cloning process. By using whole bacterial cells as carriers instead of isolating DNA into agarose plugs, the method removes the time-consuming DNA extraction, plug preparation, and embedding steps while directly transferring intact genomes (greater than 150 kb) from donor to recipient cells through cell fusion
Solution Approach 2:
The invention uses simple, inexpensive materials for cell fusion (PEG, calcium chloride, sorbitol) instead of expensive agarose plug preparation reagents. The method employs readily available reagents that can be used directly without complex preparation, significantly reducing cloning costs while maintaining the ability to transfer large genomes
3Productivity
If direct contact between donor and recipient cells is established, then transfer efficiency is improved, but incompatibility problems among different cell types arise
Solution Approach 1:
The invention uses PEG (polyethylene glycol) as an intermediary substance that mediates fusion between bacterial donor cells and yeast recipient cells. PEG creates a controlled environment that facilitates membrane fusion while protecting against incompatibility issues, allowing efficient transfer of bacterial genomes into eukaryotic yeast cells despite their different cellular structures and biological systems
Solution Approach 2:
The invention changes the physical and chemical parameters of the cell fusion environment by using PEG at specific concentrations (12-20% w/w) and controlling pH, temperature, and ionic strength. These parameter optimizations create favorable conditions for cross-species cell fusion while minimizing incompatibility problems between prokaryotic and eukaryotic cells
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 enables the efficient transfer and modification of large nucleic acids or genomes without the need for agarose plugs, reducing time and cost, and allows for the manipulation of intractable organisms by using strong genetic systems like yeast, facilitating the production of synthetic genomes and cells.
Implementation Method 1
contacting said population of bacterial donor cells with the population of yeast host cell spheroplasts in the presence of from 12% (w/w) to 20% (w/w) of a crowding agent, wherein the crowding agent is a polyethylene glycol (PEG) or Ficoll
Data Source
Figure 1a~1c
Figure 2a~2d
Figure 3a~3c
AI summary
The presently disclosed invention relates to methods of transferring large nucleic acid molecules or a genome from one cell (the donor) into heterologous host cells in the presence of a crowding agent. The method allows for greater ease and efficiency of transfer of genetic material. Introduction of the donor genetic material into the recipient host cells also allows for manipulation of the donor nucleic acid molecule or genome within the host cells. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.