Tunable Phosphorylation Feedback Controller for Mammalian Gene Expression
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Solution Overview
Problem
Current intracellular feedback control systems for gene expression in mammalian cells are not readily achievable and have limited tunability, making them susceptible to perturbations and not robust enough to maintain consistent gene expression levels.
Innovation Solution
The development of feedback controller circuits that incorporate bacterial two-component signaling systems, specifically using separate kinase and phosphatase proteins from the EnvZ-OmpR system, with a strong phosphatase regulator designed to dephosphorylate the response regulator and a destabilization domain to tune gene expression, allowing for robust regulation of output molecule expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If bacterial two-component signaling systems are incorporated into feedback controller circuits, then output molecule expression can be tuned by modulating kinase and phosphatase activity, but the system becomes more complex due to the need for separate kinase and phosphatase proteins
Solution Approach 1:
The patent divides the bifunctional histidine kinase into separate kinase and phosphatase proteins. The kinase protein (EnvZ) phosphorylates the response regulator (OmpR), while the phosphatase regulator (EnvZt) dephosphorylates it. This segmentation allows independent control of phosphorylation and dephosphorylation activities, enabling tunable gene expression without requiring a single complex bifunctional enzyme.
Solution Approach 2:
The patent introduces a phosphatase regulator (EnvZt) as an intermediary component that specifically dephosphorylates the phosphorylated response regulator. This intermediary allows the system to balance kinase and phosphatase activities independently, providing precise control over the level of phosphorylated activator and thus the tunability of output molecule expression.
2Reliability
If a strong phosphatase regulator is used to effectively dephosphorylate the response regulator, then negative regulation of transcription is enhanced, but the system becomes more sensitive to perturbations that affect phosphatase activity
Solution Approach 1:
The patent implements a feedback control mechanism where the phosphatase regulator (EnvZt) is expressed under the control of the same activable promoter as the output molecule. This creates a negative feedback loop: when output molecule expression increases, phosphatase regulator expression also increases, leading to enhanced dephosphorylation of the response regulator and subsequent reduction in transcription. This feedback mechanism maintains stable gene expression levels despite perturbations.
Solution Approach 2:
The feedback control mechanism acts as a cushioning mechanism that anticipates and compensates for perturbations. When perturbations cause off-target gene regulation or resource sequestration that reduces phosphatase regulator expression, the system responds by shifting the kinase:phosphatase balance toward kinase activity, increasing phosphorylated activator levels and compensating for the downregulation.
3Adaptability or versatility
If the phosphatase regulator is made unstable through a destabilization domain, then the degree of negative regulation can be tuned by adding or withholding a stabilizing small molecule, but the system requires additional components for stabilization
Solution Approach 1:
The patent introduces a destabilization domain (DDd) into the phosphatase regulator, making it dynamically controllable. The phosphatase regulator becomes unstable by default but can be stabilized by adding a small molecule (trimethoprim). This dynamic control allows the degree of negative regulation to be tuned in real-time by adjusting the concentration of the stabilizing small molecule, providing flexible control over gene expression levels.
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
These circuits maintain consistent output molecule expression levels, reducing sensitivity to cellular perturbations and ensuring robustness by modulating the kinase:phosphatase balance and incorporating a destabilization domain for tunable regulation.
Implementation Method 1
a kinase protein favoring phosphorylation of the response regulator
Implementation Method 2
the phosphatase regulator favoring dephosphorylation of the response regulator
Implementation Method 3
incorporation of a destabilization domain into the phosphatase regulator, which rendered the phosphatase regulator unstable unless a stabilizing small molecule was present
Data Source
AI summary
Provided herein are feedback controller circuits and cell state classifiers for tunable phosphorylation-based transcriptional regulation of gene expression in cells based on intracellular miRNA profiles and degradation by small molecules. Also provided are methods of using feedback controller circuits and cell state classifiers for determining the cell state, and methods of treating cells and subjects using feedback controller circuits and cell state classifiers encoding therapeutic output molecules.