Bacteroides Promoter and RBS Optimization for Reliable Protein Expression
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Solution Overview
Problem
There is a need for reliable genetic tools for manipulating Bacteroides, particularly for expressing nucleic acids and proteins in the human gut microbiota, as existing tools are insufficient for microscopic imaging and therapeutic applications.
Innovation Solution
Development of nucleic acids with promoters operably linked to heterologous sequences, including synthetic ribosomal binding sites, and fusion proteins for Bacteroides cells, enabling reliable expression of transgenes, labeling, and protein delivery in the gut.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing genetic tools are used for Bacteroides manipulation, then basic genetic operations can be performed, but reliable nucleic acid expression and protein secretion are insufficient
Solution Approach 1:
The patent systematically optimizes multiple parameters including promoter sequences (Pb promoters), ribosomal binding sites (RBS), and gene sequences to achieve reliable expression. Through mutational analysis and selection of specific parameter combinations, the invention identifies optimal configurations that enable consistent fluorescent protein expression and therapeutic protein secretion in Bacteroides species.
Solution Approach 2:
The patent introduces fusion proteins comprising a Bacteroides secreted polypeptide fused to a heterologous polypeptide of interest. This intermediary approach enables protein secretion by leveraging the native secretion machinery of Bacteroides while delivering therapeutic or diagnostic proteins that would otherwise not be secreted by the organism.
2Illumination intensity
If strongest available promoters are used, then some gene expression is achieved, but expression level is insufficient for microscopic imaging
Solution Approach 1:
The patent performs comprehensive optimization of promoter sequences, ribosomal binding sites, and gene sequences to achieve high-level fluorescent protein expression. Through systematic variation and selection of these parameters, the invention identifies configurations that produce sufficient fluorescence intensity for microscopic imaging while maintaining consistent expression across bacterial populations.
Data Source
AI summary
Provided are nucleic acids that include a promoter, where the promoter is operable in a Bacteroides cell and is operably linked to a heterologous nucleotide sequence of interest. Also provided are nucleic acids that include a promoter (operable in a prokaryotic cell such as a Bacteroides cell) operably linked to a sequence encoding a synthetic ribosomal binding site (RBS). Also provided are fusion proteins (and nucleic acids encoding them) in which a secreted Bacteroides polypeptide is fused to a heterologous polypeptide of interest. Also provided are prokaryotic cells (e.g., E. coli, a Bacteroides cell, and the like) that include one more nucleic acids such as those described above. Also provided are methods of expression in a prokaryotic cell, methods of detectably labeling a Bacteroides cell in an animal's gut, and methods of delivering a protein to an individual's gut.