E. coli Yeast Extract Mixture for Site-Directed Cloning
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Solution Overview
Problem
Current DNA cloning methods, such as restriction enzyme and ligase-dependent methods, face limitations including low efficiency, high costs of ligase-independent methods, and potential for mutations due to unregulated DNA repair mechanisms in bacterial extracts, making them unsuitable for small laboratories seeking cost-effective and accurate cloning solutions.
Innovation Solution
A mixture of cell extracts from wild-type E. coli and Saccharomyces cerevisiae is used for site-directed cloning, facilitating homologous recombination between the 3′ and 5′-ends of DNA fragments and linearized vectors, enabling in-frame construction of expression vectors with minimal mutations and high accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If purified enzymes are used for ligase-independent cloning methods, then cloning efficiency is improved, but cost increases significantly
Solution Approach 1:
The patent uses crude cell extracts instead of purified enzymes, replacing expensive, stable reagents with cheap, disposable cell lysates. The extracts contain all necessary recombination proteins in their natural complexity, eliminating purification costs while maintaining functional activity for cloning reactions.
Solution Approach 2:
The invention employs crude cell extracts that contain a complex mixture of proteins, enzymes, and other cellular components working together in their native environment. This composite material approach preserves the synergistic interactions present in living cells while avoiding the high costs of isolating and purifying individual enzymatic components.
2Ease of operation
If RecA-deficient E. coli extract is used for cloning, then operation is simplified, but mutation rate increases due to unregulated DNA repair
Solution Approach 1:
The patent introduces RecA protein as a controllable intermediary in the recombination process. By adding RecA to the extract in controlled amounts, the recombination reaction is facilitated while the potentially harmful unregulated DNA repair activity is prevented, as the extract comes from RecA-deficient cells rather than wild-type cells.
Solution Approach 2:
The invention changes the key parameter of RecA protein presence by using RecA-deficient E. coli strains for extract preparation, then supplementing with controlled RecA protein addition. This parameter change eliminates unregulated DNA repair mutations while preserving the ability to perform controlled homologous recombination for cloning.
3Quantity of substance
If traditional restriction enzyme and ligase methods are used, then cost is reduced, but cloning efficiency decreases due to multiple limitations
Solution Approach 1:
The patent replaces the mechanical enzymatic system of restriction enzymes and ligases with a biological recombination system based on homologous recombination. This substitution eliminates the need for precise restriction sites and ligase optimization, providing more flexible and efficient cloning while maintaining cost-effectiveness through the use of crude extracts.
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 provides a cost-effective, simple, and accurate DNA cloning process with high efficiency and minimal mutations, suitable for small laboratories, and maintains activity for extended storage periods when kept at -30°C or below.
Implementation Method 1
methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods of the '600 patents are based on homologous recombination mediated by in vitro enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.
Data Source
AI summary
The present invention provides novel reagents and a cloning procedure based on homologous recombination for the site-directed cloning of a DNA fragment to a vector at designed site(s). The cloning reagents are made of mixture of extracts from at least two different cell types, preferably a mixture made of extracts from wild-type E. coli and S. cerevisiae. Due to the activity of the mixture of cell extracts, recombination occurs between the 3′ and 5′-ends of the target DNA and at the ends of linearized vector, which facilitates in-frame construction of expression vectors.