Synthetic Operon Chromosomal Integration for Stable Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current bacterial delivery systems for biologically active agents face instability issues due to the loss of replicative plasmids over time, necessitating a stable and constitutive expression system for vaccine antigens or molecules.
Innovation Solution
A synthetic operon is constructed for integration into a bacterial chromosome, co-transcribing a gene of interest with a gene essential for the bacterium, ensuring stable expression and integration, using bacteria like Helicobacter pylori that can form chronic infections, allowing for prolonged delivery of biologically active agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If replicative plasmids are used for gene expression, then expression levels can be high, but plasmid stability deteriorates over time leading to loss of the expression system
Solution Approach 1:
The invention merges the gene of interest with an essential bacterial gene into a single operon structure that is integrated into the bacterial chromosome. This combination ensures that the gene of interest is co-transcribed with the essential gene, providing both stable inheritance (since the essential gene cannot be lost) and constitutive expression. The merging resolves the contradiction by linking the fate of the gene of interest to the essential gene, preventing plasmid loss while maintaining expression.
Solution Approach 2:
The invention segments the essential gene into two parts: the 5' portion and the 3' portion, with the gene of interest inserted between them. This segmentation allows the gene of interest to be expressed as part of the essential gene's operon while maintaining the functionality of the essential gene. The segmented structure enables stable chromosomal integration and constitutive expression without requiring separate plasmid maintenance.
2Stability of the object's composition
If plasmid stabilization methods are used, then plasmid loss is prevented, but the system requires continuous selection pressure and cannot provide truly stable expression
Solution Approach 1:
The invention makes the expression system self-sustaining by integrating it into the bacterial chromosome alongside an essential gene. The system serves itself because the essential gene's functionality is required for bacterial survival, automatically ensuring the maintenance of the gene of interest without external selection pressure. The bacterial cell itself enforces the stability by preventing loss of the integrated operon, as this would result in cell death.
Solution Approach 2:
The invention performs preliminary action by integrating the gene of interest into the bacterial chromosome before any potential plasmid loss can occur. The chromosomal integration is established upfront, creating a permanent genetic modification that eliminates the need for continuous plasmid maintenance and selection pressure throughout the bacterial lifecycle.
3Stability of the object's composition
If chromosomal integration is used, then stability is improved, but expression levels may be reduced compared to plasmid-based systems
Solution Approach 1:
The invention makes the essential gene multi-functional by having it serve both its original biological function and as a driver for expressing the gene of interest. The essential gene's promoter and transcriptional machinery are utilized to drive expression of the gene of interest, eliminating the need for separate strong promoters typically required in plasmid systems. This universal usage of the essential gene's regulatory elements maintains high expression levels while ensuring chromosomal stability.
Data Source
AI summary
The present invention relates to synthetic operons. In particular, the present invention relates to a synthetic operon for integration into a bacterial chromosome of a bacterium comprising a promoter operably-linked to at least two genes, wherein at least one gene is a gene of interest and at least one gene is a gene essential to said bacterium.