Riboregulated Feedback Promoters for Flexible Gene Expression Timing
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Solution Overview
Problem
Bacterial stress-response promoters lack flexibility in controlling gene expression timing and magnitude, limiting the dynamic regulation of metabolic pathways.
Innovation Solution
Engineered riboregulated switchable feedback promoters (rSFPs) that incorporate RNA transcriptional regulators to provide an additional layer of control over gene expression, allowing for modular activation using various mechanisms such as manual induction and quorum sensing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If bacterial stress-response promoters are used for dynamic gene regulation, then the host's natural transcriptional networks can be utilized, but the flexibility to control expression timing and overall magnitude is limited
Solution Approach 1:
The patent implements nested control by placing an RNA switch element within the promoter region, creating a hierarchical regulation system where the RNA switch (inner layer) modulates the output of the stress-response promoter (outer layer). This nested structure allows the RNA switch to fine-tune expression timing and magnitude while the promoter provides the foundational regulatory framework, thereby increasing flexibility without proportionally increasing overall system complexity.
Solution Approach 2:
The RNA switch acts as an intermediary element between the stress-response promoter and the target gene. It receives the regulatory signal from the promoter and translates it into precise control of gene expression timing and magnitude. This intermediary mechanism decouples the promoter's natural transcriptional network utilization from the desired flexible expression control, resolving the contradiction by adding a mediating layer that provides the missing flexibility.
2Manufacturing precision
If engineered riboregulated switchable feedback promoters are used, then gene expression control is enhanced, but the system complexity increases
Solution Approach 1:
The engineered promoter is segmented into distinct functional modules: the stress-response promoter region, the RNA switch element, and the target gene region. This segmentation allows each component to perform its specific function independently while contributing to the overall precise control of gene expression. The modular segmentation enables precise manipulation of expression timing and magnitude without requiring complete redesign of the entire promoter structure.
Solution Approach 2:
The RNA switch element introduces dynamic control capabilities to the promoter system, allowing it to adapt its regulatory function in response to cellular conditions. The switch can transition between different conformational states to modulate gene expression timing and magnitude dynamically. This dynamic behavior enhances manufacturing precision by enabling real-time adjustment of expression levels while maintaining a relatively simple structural framework.
Data Source
AI summary
Disclosed are systems and methods that include and utilize engineered riboregulated switchable feedback promoters (rSFPs). The disclosed systems and methods include and utilize as a component one or more expression cassettes. At least one expression cassette of the disclosed systems and methods comprises a promoter operably linked to DNA encoding an RNA switch located 3′ of the promoter and a target gene or operon located 3′ of the DNA encoding the RNA switch, where the RNA switch regulates expression of the target gene. Suitable promoters may include stress responsive promoters. The disclosed systems and methods may include and utilize a second expression cassette that includes an inducible promoter for expressing an RNA effector of the RNA switch in the first expression cassette.