Self-replicating Vector Lacking Antibiotic Resistance
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Solution Overview
Problem
The use of antibiotic resistance genes in vector construction poses risks such as environmental dissemination and transfer to pathogenic strains, limiting their use in human applications, and existing alternative selection systems require specific strain construction and defined media, making them impractical for large-scale industrial production.
Innovation Solution
A self-replicating vector devoid of antibiotic resistance genes, utilizing the ccdA/ccdB selection system, which includes a sequence encoding the ccdA protein operably linked to a promoter and the Cer locus sequence, along with a heterologous sequence linked to an inducible promoter, allowing for high expression yields without antibiotics, suitable for large volumes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotic resistance genes are used as selection markers in vector construction, then vector selection and maintenance is effective, but environmental dissemination and transfer to pathogenic strains becomes a risk
Solution Approach 1:
The invention extracts and removes the harmful antibiotic resistance gene from the vector system while maintaining the essential selection function through an alternative mechanism ( toxin-antitoxin system or auxotrophic complementation), thereby eliminating environmental risks while preserving vector control
Solution Approach 2:
The invention introduces an intermediary selection mechanism (such as a toxin-antitoxin system where the vector carries the antitoxin and the host carries the toxin, or an auxotrophic system where the vector complements a host deficiency) that mediates selection without requiring antibiotic resistance genes, thus resolving the contradiction between effective selection and environmental safety
2Object-affected harmful factors
If alternative selection systems (complementation of essential genes) are used to avoid antibiotic resistance genes, then environmental safety is improved, but construction of particular deficient strains and use of defined media is required
Solution Approach 1:
Instead of making the host cell deficient and requiring external supplementation, the invention inverts the approach by making the vector dependent on the host's essential gene product, allowing selection in standard media without complex strain construction or defined media requirements
Solution Approach 2:
The invention creates a universal selection system that works in standard media without requiring specially constructed deficient strains or defined media, making the system broadly applicable and simplifying both strain construction and routine experimentation
3Reliability
If conventional systems with antibiotic resistance genes are used, then selection is effective, but vector loss and low production efficiency occur in large volumes
Solution Approach 1:
The invention employs a selection system where the vector maintains itself through a toxin-antitoxin mechanism or auxotrophic complementation that is more stable in large-volume cultures, replacing the antibiotic-dependent system that suffers from vector loss and reduced efficiency at scale
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
This approach enables high expression yields without antibiotic use, improving confinement and production efficiency in prokaryotic cells, reducing vector loss, and increasing protein or vector production by at least 10% compared to conventional systems, while eliminating the need for antibiotics, making it suitable for industrial-scale use in humans.
Implementation Method 1
The vector according to the invention comprises as sole selection marker a sequence coding for a ccdA protein. The ccdA protein functions in association with a prokaryotic cell containing a functional gene encoding a ccdB protein.
Implementation Method 2
The gene coding for the poison (ccdB) is introduced into the bacterial chromosome of the host cell: It codes for a stable protein of approximately 100 amino acids, which binds to gyrase, inducing the death of the bacteria.
Implementation Method 3
a sequence encoding the ccdA protein operably linked to a first promoter, the Cer locus sequence and a heterologous sequence operably linked to a second promoter
Data Source
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AI summary
The subject matter of the present invention is a novel vector and the use thereof for producing a heterologous protein or a gene of interest, that can be used, for example, in the context of an immunization or gene therapy programme and concerns in particular a self-replicating vector lacking an antibiotic-resistance gene, comprising a sequence encoding the ccdA protein functionally linked to a first promoter, the sequence of the Cer locus and a heterologous sequence, functionally linked to a second promoter.