Pseudomonas Nuclease for Low-Temperature Microfluidic Flow
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Solution Overview
Problem
There is a need for alternative and improved non-specific nucleases that exhibit activity at low temperatures, such as 4°C, especially in solutions containing cation complexing agents like EDTA, and at neutral to basic pH, to prevent viscosity increases and clogging in cell culture solutions.
Innovation Solution
Identification of a non-specific nuclease from Pseudomonas syringae (P.vNuc) with variants showing high catalytic rate constants and tolerance to EDTA, allowing activity across a range of temperatures and pH values, including 4°C and neutral to basic pH conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If non-specific nucleases are used to degrade nucleic acids, then viscosity increase and clogging are prevented, but the enzymes lose activity in the presence of EDTA and at low temperatures
Solution Approach 1:
The patent identifies and characterizes non-specific nucleases from Pseudomonas syringae that have been adapted to function optimally at low temperatures (4°C) and remain active in the presence of EDTA. This involves selecting enzyme variants with modified kinetic parameters that allow them to maintain catalytic activity under these specific conditions, thereby resolving the contradiction between reliability and sensitivity to harmful factors.
2Stability of the object's composition
If cell culture solutions are maintained at low temperatures to minimize cell proliferation and activation, then cell stability is improved, but nucleic acid degradation is inhibited
Solution Approach 1:
The patent employs nucleases with modified temperature-optimum parameters that are adapted to function at low temperatures (4°C). These enzymes have been selected or engineered to maintain catalytic activity at temperatures where conventional nucleases are inactive, thus enabling nucleic acid degradation without compromising cell stability.
3Ease of operation
If cation complexing agents like EDTA are added to prevent cell aggregation, then cell dispersion is improved, but nuclease activity is inhibited
Solution Approach 1:
The patent identifies nucleases from P. syringae that have been adapted to function in the presence of EDTA and other cation complexing agents. These enzymes have modified metal-ion dependence characteristics, allowing them to maintain activity despite the chelating effect of EDTA, thus resolving the contradiction between ease of operation and reliability.
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 P.vNuc enzyme effectively degrades nucleic acids, reducing solution viscosity and preventing clogging in cell culture and sorting devices, maintaining activity in the presence of EDTA and varying temperatures.
Implementation Method 1
Nucleases are nucleic acids-degrading enzymes involved in natural processes including replication, recombination, repair, and restriction
Data Source
AI summary
The present invention provides a composition comprising an isolated polypeptide having non-specific nuclease (NSN) activity for degrading nucleic acids comprising or consisting of at least 70% amino acid sequence identity to SEQ ID NO:2, wherein said polypeptide has NSN activity in a solution of about 4° C., and a cation complexing agent such as EDTA. Said composition may have preferentially a neutral to basic pH. A kit comprising said composition and a method using said composition are also disclosed.