Cell-Free Protein Synthesis with DNA Ratio Control for Co-Expression
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Solution Overview
Problem
Existing methods fail to quantitatively co-express multiple proteins in vitro using cell-free protein synthesis systems, limiting their application in research and development of fluorescently-labeled cells or organisms.
Innovation Solution
A method involving establishing a standard curve for luminescence values, creating separate vectors for each protein, and adjusting vector concentrations to achieve quantitative co-expression of multiple proteins in an in vitro cell-free protein synthesis system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cell-free protein synthesis system is used to express multiple proteins, then expression speed and convenience are improved, but quantitative control of protein expression ratios is lost
Solution Approach 1:
The patent changes the concentration parameters of template DNA for different proteins in the cell-free synthesis system. By adjusting the template DNA concentrations to specific ratios (e.g., 1:1, 1:2, 2:1), the system achieves quantitative control over protein expression ratios while maintaining the speed advantages of cell-free synthesis. This parameter adjustment allows precise control without sacrificing productivity.
2Adaptability or versatility
If multiple fluorescent proteins are co-expressed in vitro, then research applications are expanded, but measurement and detection accuracy deteriorates due to spectral overlap
Solution Approach 1:
The patent segments the detection process by using sequential acquisition modes with appropriate filters for different fluorescent proteins. This allows separate measurement of each protein's fluorescence signal, eliminating interference from other proteins in the mixture. The segmentation approach enables accurate quantification of multiple proteins simultaneously expressed in the same reaction system.
Solution Approach 2:
The patent employs fluorescence intensity measurements as feedback to verify the actual expression ratios of co-expressed proteins. By measuring the fluorescence of each protein and comparing against expected ratios, researchers can confirm whether the template DNA concentration ratios achieved the desired protein expression ratios, enabling iterative optimization.
3Reliability
If traditional in-cell protein engineering is used, then biological function context is maintained, but process complexity and time consumption increase
Solution Approach 1:
The patent extracts the protein synthesis process from living cells to a cell-free system. This extraction removes the complexity of cell growth, transformation, and cultural conditions while maintaining the ability to express functional proteins. The cell-free system uses purified components (extracts, substrates, enzymes) to synthesize proteins without requiring intact cellular machinery, dramatically simplifying the engineering process.
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
Enables efficient and intuitive real-time monitoring of protein expression, allowing for the synthesis of multiple proteins at preset ratios, particularly fluorescent proteins, with high accuracy and simplicity.
Implementation Method 1
The cell-free protein synthesis system involves an emerging technology-- cell-free synthesis system, also known as in vitro synthesis system, comprising in vitro transcription and translation
Implementation Method 2
The cell-free protein synthesis system involves an emerging technology-- cell-free synthesis system, also known as in vitro synthesis system, comprising in vitro transcription and translation
Implementation Method 3
Fluorescent proteins have been widely used in many research fieldsof biology. Fluorescent protein-based molecular probes and labeling methods have become important research tools for studying biological macromolecules or cell functions through dynamic imaging in living cells or in vivo
Data Source
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AI summary
Provided is a method for quantitatively co-expressing multiple proteins in an in vitro cell-free protein synthesis system, comprising the following steps: (1) establishing a standard curve of the relationship between standard protein concentration and luminescence value; (2) creating a vector containing a target protein gene, and obtaining an in vitro protein synthesis system; (3) establishing a curve of the relationship between the target protein concentration and the vector concentration; (4) calculating the concentration or/and the concentration ratio of the vector quantitatively co-expressing multiple target proteins; (5) quantitatively co-expressing the target proteins.