Self-deleting Plasmid via Site-specific Recombinase Excision
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Solution Overview
Problem
Current plasmid selection systems in biotechnology rely on antibiotic resistance genes, which impose a metabolic burden on host cells, contaminate products, and risk antibiotic resistance in pathogens, necessitating a marker-free selection method that does not require genetic modification of host cells.
Innovation Solution
A method involving site-specific recombinase target sites and endogenous recombinases to excise selectable marker genes from plasmids, allowing plasmid maintenance without a plasmid maintenance system, by altering host cell environments to enable or inhibit recombination between these sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotic resistance genes are used for plasmid selection, then plasmid maintenance is achieved, but metabolic burden increases and cell viability decreases
Solution Approach 1:
The patent removes the harmful antibiotic resistance gene from the plasmid system while maintaining plasmid stability through alternative mechanisms. The selectable marker gene is excised after initial selection, leaving a clean plasmid that maintains reliability without the metabolic burden of continuous antibiotic resistance gene expression.
Solution Approach 2:
The patent performs selection using antibiotic resistance genes temporarily during the initial plasmid introduction phase, then removes these genes before production. This preliminary selection action ensures plasmid maintenance during critical early stages, followed by gene excision to eliminate ongoing metabolic burden.
2Measurement precision
If antibiotic resistance genes are used for plasmid selection, then plasmid identification is enabled, but antibiotic contamination occurs in final products
Solution Approach 1:
The patent extracts and removes the antibiotic resistance gene after it has served its identification purpose during transformation and selection. This ensures that the final plasmid product is free from antibiotic contamination while having already fulfilled its identification function.
Solution Approach 2:
The antibiotic resistance gene performs its identification function during preliminary selection steps, then is removed before final product formation. This timing ensures accurate plasmid identification without carrying forward antibiotic contamination into the final product.
3Reliability
If antibiotic resistance genes are used for plasmid selection, then selective pressure is maintained, but antibiotic degradation reduces selection effectiveness
Solution Approach 1:
The patent concentrates the selection pressure action into the initial transformation phase where antibiotic resistance genes are temporarily expressed. After this preliminary selection establishes the desired plasmid population, the resistance genes are removed and selection is no longer needed, avoiding the problem of antibiotic degradation over extended periods.
4Reliability
If antibiotic resistance genes are used in live bacterial vectors, then plasmid selection is achieved, but transfer to environmental pathogens creates antibiotic resistant strains
Solution Approach 1:
The patent removes the antibiotic resistance gene from live bacterial vectors after initial selection, eliminating the risk factor that could be transferred to environmental pathogens. The gene is extracted once selection is complete, leaving a safe vector that can be deployed without creating antibiotic resistant environmental strains.
Solution Approach 2:
The antibiotic resistance gene performs its protective selection function during the controlled laboratory phase of vector development, then is removed before the vector is released into environmental or clinical settings. This preliminary protection followed by removal ensures safe deployment without long-term antibiotic resistance risks.
5Reliability
If selectable marker genes are expressed constitutively, then plasmid selection is maintained, but cell viability and growth are reduced
Solution Approach 1:
The selectable marker gene is expressed constitutively only during the preliminary selection phase to establish plasmid-containing cells. After selection is achieved, the gene is removed via excision, eliminating the ongoing burden on cell growth and productivity while maintaining the plasmid selection that was established during the preliminary phase.
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 reduces metabolic burden, eliminates antibiotic contamination, and avoids genetic modification of host cells, enabling stable maintenance and production of recombinant proteins and DNA vaccines without selectable marker genes.
Implementation Method 1
a plasmid containing a selectable marker gene flanked by site specific recombinase target sites
Data Source
AI summary
A method of producing a selectable marker gene-free plasmid by culturing a plasmid containing a selectable marker gene flanked by site specific recombinase target sites in a host cell environment incapable of effecting recombination between the site specific recombinase target sites and subsequently culturing the plasmid in another host cell environment which is capable of effecting recombination between the site specific recombinase target sites, so that the selectable marker gene is excised. Uses of plasmids produced by the method for the production of recombinant protein for therapeutic and vaccine purposes, production of therapeutic DNA and DNA vaccines and delivery of recombinant protein and DNA to a patient using live bacterial vectors.