Competent Cell Storage Buffer for -20°C Stability
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Solution Overview
Problem
Current methods for storing competent bacterial cells at -80°C are costly and result in significant loss of transformation efficiency when thawed, making them unavailable for DNA uptake.
Innovation Solution
Storing competent prokaryotic cells, such as E. coli, in a buffer containing glucose or oligosaccharides like trehalose or sucrose without salt at -20°C, and adding a salt-containing buffer proximate to transformation, with optional inclusion of glycerol and DMSO, to maintain viability and transformation efficiency for extended periods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If competent cells are stored at -80°C in high salt buffer, then storage stability is improved, but transformation efficiency is lost and cost increases
Solution Approach 1:
The patent changes the storage parameters by removing salt from the buffer and using -20°C instead of -80°C, while adding glucose or oligosaccharides. This parameter modification allows cells to maintain transformation efficiency without requiring costly -80°C storage, resolving the contradiction between storage stability and transformation efficiency
Solution Approach 2:
The patent replaces expensive long-term storage at -80°C with a cheaper alternative using glucose/oligosaccharide buffers at -20°C. The cells can be stored for extended periods (5+ days) at this lower cost, making the storage solution more economical while maintaining functionality
2Duration of action of stationary object
If competent cells are stored at -80°C, then storage duration is extended, but transformation efficiency is significantly reduced
Solution Approach 1:
By changing the buffer composition to exclude salt and include glucose or oligosaccharides, and adjusting temperature to -20°C, the patent enables extended storage (5+ days) while preserving transformation efficiency. This parameter change allows the cells to remain competent without the efficiency loss associated with conventional -80°C storage
3Stability of the object's composition
If salt-containing buffer is used for storage, then cell competency is maintained, but cells become unavailable for DNA uptake after thawing
Solution Approach 1:
The patent extracts salt from the storage buffer, using glucose or oligosaccharides instead. This removal of salt prevents the cells from becoming unavailable for DNA uptake after thawing, while still maintaining cell competency through the alternative buffer components
Solution Approach 2:
The patent introduces glucose or oligosaccharides as intermediary substances that mediate between the removal of salt and the maintenance of cell competency. These intermediaries allow cells to remain competent and available for DNA uptake without requiring salt in the storage buffer
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 allows for sustained transformation efficiency of bacterial cells stored at -20°C for up to 105 days with minimal loss, compared to standard storage at -80°C, enabling prolonged availability for DNA uptake.
Implementation Method 1
storing cells at -20° C. for a period of time
Implementation Method 2
adding a salt containing buffer to the cells at a time proximate to transformation of the cells with a foreign molecule
Data Source
AI summary
Compositions and methods are provided for storing prokaryotic cells including competent prokaryotic cells at −20° C. in a buffer so that the cells are suitable for transformation at 0° C. with a foreign molecule.