Bacteroides Genetic Circuit Tools for Microbiome Engineering
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Solution Overview
Problem
There is a lack of genetic parts and inducible systems available for Bacteroides species like B. thetaiotaomicron, which hinders the precise engineering of these bacteria for microbiome applications due to unique promoter and RBS architectures, making it difficult to incorporate genetic systems developed in other organisms.
Innovation Solution
A library of constitutive and inducible promoters, ribosome-binding sites (RBS), and recombinase-based memory gates are developed to achieve a wide range of gene expression and enable the manipulation of gene expression in Bacteroides and Parabacteroides species, including the use of CRISPR interference for regulated knockdown of genes and construction of synthetic genetic memory.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If genetic systems from other organisms are used in Bacteroides, then gene expression control is achieved, but compatibility and precision are poor due to unique promoter and RBS architectures
Solution Approach 1:
The patent systematically varies promoter sequences and RBS sequences to create libraries with different expression strengths. By changing nucleotide parameters in promoter regions (e.g., -35 and -10 box sequences) and RBS regions (e.g., Shine-Dalgarno sequence variations), the invention achieves precise control over gene expression levels while maintaining compatibility with Bacteroides native transcription and translation machinery
Solution Approach 2:
The patent creates specific genetic parts optimized for Bacteroides by analyzing and replicating native Bacteroides promoter and RBS sequences. Instead of using generic bacterial parts, the invention designs local genetic elements with sequences matching Bacteroides characteristics, ensuring proper recognition by Bacteroides RNA polymerase and ribosomes while achieving desired expression levels
2Adaptability or versatility
If a library of promoters and RBS is created to achieve wide expression range, then gene expression control is improved, but device complexity increases
Solution Approach 1:
The patent creates universal genetic parts that can be combined in various configurations to achieve different expression levels. The promoter library and RBS library are designed as modular, interchangeable components that work together in predictable ways, allowing researchers to mix and match parts to achieve desired expression ranges without designing entirely new systems for each application
Solution Approach 2:
The patent divides the gene expression control system into separate, independent modules: promoter regions, RBS regions, and coding sequences. Each module can be independently selected and optimized, then assembled together. This segmentation allows the complexity to be managed through modular design, where the overall expression range is achieved through combinations of standardized parts rather than a single complex element
Data Source
AI summary
Provided herein, in some aspects, are tools (e.g., methods, compositions and nucleic acids) for building genetic circuits in Bacteroides and Parabacteroides bacteria, as well as the bacteria containing the genetic circuits.