Cell Free Synthetic Biology System for Rapid Production of Diagnostic Biomolecules

Abstract

Abstract: This invention provides a cell-free synthetic biology platform for rapid and scalable production of diagnostic biomolecules, such as proteins, antibodies, enzymes, and nucleic acids. The system utilizes optimized reaction conditions and engineered biosynthetic pathways to synthesize high-quality biomolecules outside of living cells, providing a rapid and flexible alternative to traditional cell-based production methods. This invention is particularly useful for developing diagnostics for pathogens, disease biomarkers, and genetic sequences, and enables high-throughput screening, customized production, and point-of-care testing applications.

Specification

Description:Background of the Invention:
Diagnostic biomolecules, such as enzymes, antibodies, and nucleic acids, are essential components in disease detection, point-of-care diagnostics, and biosensing applications. Conventional methods for producing these biomolecules involve cell-based systems, which are time-consuming, limited by cell viability, and difficult to customize for rapid responses to emerging pathogens or biomarkers. Cell-free synthetic biology provides a promising alternative, offering faster and more flexible production of diagnostic biomolecules. However, current cell-free systems face challenges in efficiency, yield, and scalability, necessitating improvements in reaction conditions, cofactor regeneration, and pathway optimization.
This invention addresses these limitations by presenting a cell-free system designed for the efficient and rapid synthesis of high-quality diagnostic biomolecules with optimized production of functional proteins, nucleic acids, and other biomolecules used in diagnostic assays.
Summary of the Invention:
The present invention provides a cell-free synthetic biology system designed for the high-yield, rapid production of diagnostic biomolecules, such as antibodies, nucleic acid probes, and enzymes used in diagnostic applications. Key innovations include:
1. Optimized Reaction Conditions for Biomolecule Synthesis:
o Optimized buffer systems and cofactors that enhance reaction stability, yield, and efficiency of biomolecule synthesis.
o Use of tailored amino acid mixtures and energy regeneration systems to extend the lifespan and productivity of cell-free reactions.
2. Gene Expression Systems for Rapid Protein Synthesis:
o Incorporation of inducible and high-efficiency promoters for protein synthesis, allowing the selective expression of diagnostic proteins.
o Use of cell-free transcription-translation (TX-TL) systems for rapid synthesis of diagnostic enzymes and antibody fragments.
3. Efficient Cofactor Recycling and Energy Regeneration:
o Systems to recycle ATP and NADPH to sustain high-energy-demand processes, improving reaction efficiency and biomolecule yield.
o Engineering of novel cofactors and regeneration enzymes to support prolonged reaction times without external addition of substrates.
4. Integration of Modular Biosynthetic Pathways for Customizable Production:
o Modular pathway assemblies that allow for on-demand synthesis of various biomolecules, adaptable to different diagnostic needs.
o Use of interchangeable genetic modules for rapid switching between different biomolecule targets, supporting flexible diagnostic applications.
5. High-Throughput Screening and Automation Capabilities:
o Integration of the system with automated platforms to enable high-throughput production and screening of diagnostic biomolecules.
o Real-time monitoring of reaction conditions using biosensors to optimize and control product output.
6. Scalability for Point-of-Care and On-Site Production:
o Portable, miniaturized versions of the cell-free system designed for point-of-care applications, enabling on-site biomolecule production.
o Low-cost, scalable reaction setups suitable for rapid deployment in resource-limited settings or outbreak responses.
Detailed Description of the Invention:
1. Optimized Reaction Conditions for Cell-Free Biosynthesis:
The invention provides a specially formulated reaction buffer, enriched with essential cofactors, salts, and stabilizing agents, to maintain high stability and activity throughout the synthesis process. The buffer is optimized for sustained synthesis in both transcription and translation, enabling the production of structurally complex biomolecules, such as full-length antibodies and multimeric proteins, which are critical for diagnostics.
The reaction conditions also feature optimized concentrations of magnesium ions, ATP, and other critical cofactors that increase the overall productivity and reduce the cost of production by enabling longer reaction times without frequent additions of external components.
2. High-Efficiency Gene Expression System:
A cell-free transcription-translation (TX-TL) platform is used to rapidly express genetic constructs encoding diagnostic biomolecules. The system incorporates inducible promoters and regulatory elements to control protein expression levels. Using in vitro synthesized mRNA or plasmid DNA, the platform achieves high yields of diagnostic enzymes, antibody fragments, and nucleic acid binding proteins.
For example, a DNA template encoding a pathogen-specific antibody fragment can be transcribed and translated within hours, producing high-purity diagnostic reagents in a fraction of the time required by traditional cell-based systems.
3. Enhanced Cofactor Recycling System:
The invention employs a novel cofactor recycling system, incorporating enzymes such as phosphoenolpyruvate (PEP) and NADH oxidase to regenerate ATP and NADPH within the reaction. This recycling system sustains the high-energy demands of the cell-free system, enabling prolonged synthesis and reducing the cost associated with frequent cofactor supplementation.
This aspect is critical for diagnostics as it provides stable reaction conditions, ensuring a continuous production of active biomolecules without the need for external refueling, thus enhancing reliability and usability for end-users.
4. Modular Pathway Integration for Customizable Production:
The invention incorporates a modular design where interchangeable gene cassettes encoding various biomolecules can be easily swapped within the system. Each module contains regulatory elements tailored to the specific expression needs of different biomolecule types (e.g., enzymes vs. antibodies), allowing for versatile diagnostic applications. For instance, diagnostic applications requiring the detection of different pathogens can rapidly switch between DNA templates encoding pathogen-specific antibodies or nucleic acid probes.
5. Automation and High-Throughput Production:
To meet the demand for high-throughput production of diagnostic biomolecules, the cell-free system is compatible with automated platforms that support rapid screening and optimization of biomolecule production. Automated controls monitor parameters such as pH, temperature, and reaction progress, providing real-time data to optimize production yield and quality.
6. Point-of-Care and On-Site Production Capabilities:
The invention includes a miniaturized, portable version of the cell-free system, allowing for on-site production of diagnostic biomolecules in settings such as clinics, field hospitals, and remote areas. This capability supports point-of-care diagnostics, enabling healthcare providers to produce and deploy diagnostic reagents on-demand in low-resource or emergency environments.
, Claims:Claims:
1. A cell-free system for producing diagnostic biomolecules comprising an optimized reaction buffer, high-efficiency gene expression system, and a cofactor recycling system.
2. The system of claim 1, wherein the gene expression system uses inducible promoters for controlled production of target proteins, enzymes, or antibodies.
3. A method for producing diagnostic biomolecules using a modular cell-free platform, wherein DNA templates encoding different biomolecules can be interchanged for customizable production.
4. The system of claim 3, further comprising high-throughput screening capability with real-time monitoring of reaction conditions.
5. A portable, miniaturized version of the system for on-site production of diagnostic biomolecules, suitable for point-of-care applications in remote or resource-limited settings.

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