Escherichia coli Trehalose Accumulation for Competent Cell Storage
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Solution Overview
Problem
Current methods for preparing Escherichia coli competent cells require storage at low temperatures to maintain transformation efficiency, which is energy-intensive and costly, and involve the use of additional anti-freezing agents.
Innovation Solution
A bacterial strain of Escherichia coli capable of spontaneously accumulating self-producing trehalose is used, allowing competent cells to be stored at higher temperatures without a significant decrease in transformation efficiency, eliminating the need for additional anti-freezing agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If competent cells are stored at low temperatures (−70°C to −80°C), then transformation efficiency is maintained, but energy consumption increases and storage cost rises
Solution Approach 1:
The invention changes the chemical composition parameter of the storage medium by adding specific anti-freezing agents (such as glycerol, ethylene glycol, or sugars) to enable competent cells to be stored at higher temperatures (−20°C to 4°C) while maintaining transformation efficiency. This parameter change in storage temperature directly reduces energy consumption for refrigeration while preserving the reliability of transformation efficiency.
2Temperature
If additional anti-freezing agents are added to storage medium, then competent cells can be stored at higher temperatures, but preparation cost increases
Solution Approach 1:
The invention utilizes substances that are either naturally present in the competent cell system or can be produced through simple metabolic processes of the cells themselves. For example, certain bacteria can produce trehalose or other protective sugars endogenously when exposed to cold stress, eliminating or reducing the need for externally added anti-freezing agents. This self-service approach raises storage temperature capability while controlling preparation costs.
3Reliability
If conventional chemical transformation process is used, then transformation efficiency can be improved, but storage requirements become more stringent
Solution Approach 1:
The invention creates a composite storage system that combines chemical transformation agents (such as calcium chloride, RbCl, or CsCl) with physical protective elements (such as frozen droplets, protective proteins, or cell wall modifications). This composite approach allows the competent cells to maintain high transformation efficiency from the chemical process while the physical protective elements enable more relaxed storage temperature requirements.
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
The method enables stable transformation efficiency at higher storage temperatures, reducing energy costs and simplifying the preparation of competent cells while maintaining high transformation efficiency, reaching up to 10^9 transformants/μg plasmid DNA.
Implementation Method 1
the cell is capable of spontaneously accumulating self-producing trehalose therein
Data Source
AI summary
The invention provides a cell for preparing competent cells, wherein the cell is capable of spontaneously accumulating self-producing trehalose therein and the cell is used for the preparation of competent cells. The invention also provides a method for preparing competent cells, including: culturing the cell for preparing the competent cell mentioned previously to obtain a cell suspension; placing the cell suspension into an ice bath; centrifuging the cell suspension to obtain a cell precipitate; mixing a transform reagent with the cell precipitate; and obtaining competent cells suspension.