Split-Intein Genetic Controllers for Constant Protein Output
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Solution Overview
Problem
Existing genetic controllers struggle to maintain a constant-level concentration of output proteins due to limitations in design and compatibility across different host organisms, particularly in bacteria and eukaryotes, and lack the robustness to adapt to varying conditions and disturbances.
Innovation Solution
Utilizing split-inteins to create genetic controllers capable of achieving Robust Perfect Adaptation (RPA) by implementing an antithetic integral feedback motif, which involves inserting split-inteins into transcription factors or other proteins to achieve irreversible protein splicing and stoichiometric sequestration, ensuring the expression system maintains a constant output level despite perturbations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional genetic controllers are used, then the system can express output proteins, but the concentration level cannot be maintained constant despite disturbances
Solution Approach 1:
The patent implements an antithetic integral feedback controller where the controller protein Z1 and anti-controller protein Z2 dynamically adjust their concentrations based on feedback from the output protein X. The controller activates the output, which in turn activates the anti-controller, creating a negative feedback loop that maintains constant output concentration despite external disturbances or internal parameter changes.
Solution Approach 2:
The system changes the concentration parameters of the controller and anti-controller proteins dynamically. When the output concentration increases, the anti-controller concentration increases accordingly, altering the feedback parameter to bring the output back to the setpoint. This dynamic parameter adjustment enables robust adaptation to varying conditions.
2Reliability
If integral feedback control is implemented to achieve Robust Perfect Adaptation, then the output concentration can be maintained constant, but the device complexity increases
Solution Approach 1:
The feedback controller is segmented into two distinct functional components: the controller protein Z1 that activates the output, and the anti-controller protein Z2 that deactivates the output. This segmentation allows each component to have specialized functions, simplifying the overall design while achieving robust perfect adaptation through their interactive feedback loop.
Solution Approach 2:
The output protein X serves as an intermediary that mediates between the controller and anti-controller. It activates both the controller (positive feedback) and the anti-controller (negative feedback), enabling the system to achieve robust adaptation without requiring complex direct interaction mechanisms between the controller and anti-controller themselves.
3Reliability
If split-inteins are used to achieve irreversible protein splicing, then stoichiometric sequestration is achieved, but the manufacturing precision requirements increase
Solution Approach 1:
The split-intein system performs self-service through autocatalytic protein splicing. The intein segments automatically recognize and splice with their complementary partners without requiring external enzymatic intervention or precise manual assembly. This self-service mechanism achieves stoichiometric sequestration while reducing manufacturing precision requirements, as the splicing reaction occurs naturally upon mixing the split-intein components.
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
The split-intein-based controllers ensure robust and efficient maintenance of constant protein expression levels by leveraging orthogonal pairs and minimal cellular burden, enhancing dynamic performance and adaptability under stochastic conditions.
Implementation Method 1
An intein is a segment of a protein that is capable of autocatalytically cutting itself from the protein and reconnecting the remaining segments, called exteins, via peptide bonds
Implementation Method 2
These small protein segments are capable of heterodimerizing and performing protein splicing reactions on their own where they break and form new peptide bonds
Data Source
AI summary
The present invention relates to an expression system and a method ensuring constant-level concentration of an output. The invention also relates to a cell comprising the expression system.