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Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells
Published on: July 6, 2021
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Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells
Michael Tyler Guinn1, Damiano Coraci2, Lesia Guinn2
1Biomedical Engineering Department, Stony Brook University; Laufer Center for Physical and Quantitative Biology, Stony Brook University; Stony Brook Medical Scientist Training Program; michael.guinn@stonybrookmedicine.edu.
Journal of Visualized Experiments : Jove
|July 26, 2021
Summary
This study presents a standardized pipeline for building and testing optogenetic gene expression systems in mammalian cells. This approach enables precise control over gene expression, reducing noise and improving accuracy for various applications.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Biotechnology
Background:
- Optogenetic systems offer spatiotemporal control of gene expression in mammalian cells.
- Existing optogenetic circuits often lack standardization in architecture, expression methods, and equipment.
- There is a need for characterized optogenetic components within stable cell lines.
Purpose of the Study:
- To develop and validate an experimental pipeline for constructing, integrating, and characterizing optogenetic gene circuits.
- To demonstrate the utility of standardization in optogenetic equipment and light conditions.
- To provide a framework for reliable gene expression control in mammalian cells.
Main Methods:
- Development of a standardized pipeline for gene circuit construction and integration.
- Utilization of a negative feedback optogenetic circuit as a model system.
- Characterization of gene expression noise and protein output magnitude under standardized conditions.
Main Results:
- The pipeline enables reliable construction and characterization of light-inducible gene expression circuits.
- Standardized protocols reveal key gene circuit features like noise and expression levels.
- The developed system allows for precise control over transcriptional, proteomic, and phenotypic outputs.
Conclusions:
- The presented pipeline facilitates the adoption of optogenetics by laboratories unfamiliar with the technology.
- Standardization of optogenetic components and protocols is crucial for reproducible gene expression control.
- This approach enhances the reliability and detail of gene expression characterization in mammalian cells.
