Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles

Area of Science:

• Synthetic biology
• Cellular engineering
• Biochemical signaling

Background:

• Engineered signaling networks are crucial for cellular therapies and directing differentiation.
• Existing networks often lack tunability, precise control, and robustness in mammalian cells.

Purpose of the Study:

• To develop tunable, precise, and robust synthetic phosphoregulation devices in mammalian cells.
• To create cell-type specific signaling responses and a method for cell classification.
• To implement a negative feedback controller for stable gene expression.

Main Methods:

• Utilized bacterial two-component signaling proteins, specifically the EnvZ/OmpR system.
• Engineered a synthetic covalent modification cycle with kinase and phosphatase activities.
• Regulated phosphatase expression using endogenous microRNAs (miRNAs).
• Implemented a small molecule-stabilized phosphatase for negative feedback control.

Main Results:

• Achieved analog tuning of gene expression via the EnvZ/OmpR pathway.
• Demonstrated cell-type specific signaling responses by regulating phosphatase with miRNAs.
• Developed a new strategy for accurate cell type classification.
• Reduced output expression variance and mitigated off-target effects using negative feedback.

Conclusions:

• Synthetic phosphoregulation devices based on bacterial signaling proteins offer tunable, precise, and robust control in mammalian cells.
• This technology provides a foundation for advanced cellular therapies and synthetic biology applications.
• The developed system enables sophisticated control over cellular behavior and gene expression.