Tunable Synthetic Protein Circuits for Signal Thresholding
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Solution Overview
Problem
Existing synthetic biology approaches lack efficient mechanisms for designing protein circuits that respond only above or below a certain threshold concentration, limiting the functionality and precision of protein-level circuits in living cells.
Innovation Solution
Development of synthetic protein circuits comprising specific polypeptide domains that associate or dissociate based on proximity and binding affinity, allowing for thresholding outputs and modulating protein activity, stability, and localization through tuner polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If synthetic biology approaches use conventional gene regulation circuits, then the design of new functions in living cells is enabled, but the precision and functionality of protein-level circuits are limited
Solution Approach 1:
The invention divides the protein circuit into distinct modular components: input polypeptides with partner domains, output polypeptides, and thresholding polypeptides. Each module performs a specific function (signal reception, signal transmission, threshold determination), allowing independent optimization and precise control of threshold responses through component composition and stoichiometry.
Solution Approach 2:
The invention enables precise threshold control by varying parameters such as the stoichiometric ratios of input polypeptides to thresholding polypeptides, the binding affinity of partner domains, and the concentration of thresholding polypeptides. These parameter changes allow tuning of the threshold concentration without altering the fundamental circuit architecture.
2Measurement precision
If synthetic protein circuits are designed to respond only above or below certain threshold concentrations, then the precision of protein activity control is improved, but the device complexity increases
Solution Approach 1:
The invention employs universal partner domains that can mediate binding between different polypeptide pairs, allowing the same binding interface to be reused across multiple circuit components. This multi-functionality reduces the need for unique interaction domains for each component, thereby simplifying the overall circuit design while maintaining precise threshold control.
Solution Approach 2:
The thresholding polypeptide acts as an intermediary component that receives input signals through partner domain binding and translates them into threshold-dependent output responses. This mediator component simplifies the circuit architecture by centralizing the threshold determination function, avoiding the need for complex regulatory networks.
3Measurement precision
If polypeptide domains are designed with specific binding affinities for thresholding, then the activation precision is improved, but the difficulty of detecting and measuring binding interactions increases
Solution Approach 1:
The invention incorporates fluorescent or colorimetric reporters that change their optical properties upon binding or proximity events. These optical changes provide direct, quantifiable readouts of binding affinity and threshold activation, transforming difficult-to-measure protein-protein interactions into easily detectable optical signals.
Solution Approach 2:
The invention replaces direct mechanical or biochemical measurement of binding interactions with optical detection methods. By coupling binding events to fluorescent resonance energy transfer (FRET) or other optical reporters, the system substitutes complex biophysical measurements with simpler, more precise optical readings.
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 precise control of protein activity and output levels by ensuring activation only above or below defined input concentrations, enhancing the functionality and precision of protein circuits in living cells.
Implementation Method 1
the first partner domain is capable of binding the second partner domain, wherein the first polypeptide domain and the second polypeptide domain are capable of associating with each other to constitute a first protein capable of being in a first protein active state when the first partner domain binds the second partner domain
Implementation Method 2
the third partner domain is capable of binding the second partner domain, wherein the first protein is not in the first protein active state when the third partner domain binds the second partner domain
Implementation Method 3
synthetic protein circuits comprising specific polypeptide domains that associate or dissociate based on proximity and binding affinity, allowing for thresholding outputs and modulating protein activity, stability, and localization through tuner polypeptides
Data Source
AI summary
Disclosed herein include methods, compositions, and kits suitable for use in thresholding of protein signals. There are provided, in some embodiments, synthetic protein circuits that respond to inputs only above or below a certain threshold concentration. In some embodiments, the threshold value itself is tunable. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.