Antibiotic-Free Marker Composition for Living Modified Organisms
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Solution Overview
Problem
Current methods for producing living modified organisms (LMOs) rely on antibiotic resistance markers, which increase production costs, cause environmental pollution, and lead to antibiotic resistance mutations, and are inefficient due to antibiotic degradation and instability, limiting the use of antibiotic-free systems in industrial bioprocesses.
Innovation Solution
A marker composition and transformation method using plasmids with genes encoding enzymes in the isopentenyl diphosphate or dimethylallyl diphosphate synthetic pathway, allowing for antibiotic-free selection and maintenance of modified organisms by activating or complementing these pathways, thereby avoiding the use of antibiotic resistance genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If antibiotic resistance markers are used for selection, then transformation efficiency is improved, but production costs increase and environmental pollution occurs
Solution Approach 1:
The patent removes antibiotic resistance markers from the transformation system entirely, extracting the harmful selection method while retaining transformation efficiency through auxotrophic selection using complementation plasmids that restore essential metabolic pathways
Solution Approach 2:
The patent introduces auxotrophic markers as an intermediary selection mechanism, where essential metabolic genes (dxs, dxr, ispD, ispE, ispF, or ispG) serve as selectable markers instead of antibiotic resistance genes, enabling selection without antibiotics
2Stability of the object's composition
If antibiotics are used during culture, then plasmid stability is maintained, but antibiotic degradation occurs and productivity decreases
Solution Approach 1:
The patent employs auxotrophic selection where the organism's own metabolic requirements drive plasmid maintenance - the plasmid carries essential genes that the host cannot produce independently, creating self-enforcing plasmid retention without external antibiotics
Solution Approach 2:
The patent eliminates antibiotics from the culture system, removing the source of degradation and modification that plasmid-encoded enzymes cause, thereby maintaining plasmid stability through biological selection rather than chemical pressure
3Object-generated harmful factors
If foreign genes are inserted into chromosomes, then antibiotic marker-free selection is achieved, but gene expression amount decreases
Solution Approach 1:
The patent maintains plasmids with high copy numbers in the cytoplasm rather than integrating into chromosomal DNA, preserving local high-concentration gene expression while using auxotrophic markers for selection instead of antibiotics
Solution Approach 2:
The patent uses auxotrophic markers as an intermediary selection system that operates independently of chromosomal integration, allowing plasmid-borne genes to be selected and maintained without antibiotic pressure while preserving high expression levels
Data Source
AI summary
The present invention provides a marker composition for selecting a living modified organism, which allows transformation and the production of a target product without antibiotics or antibiotic resistance genes, a living modified organism, a method of transforming an organism, and a method of producing a target product. The present invention provides a marker composition for selecting a living modified organism, which may basically prevent problems caused by the use of antibiotics and antibiotic resistance genes and produce a target product at a high yield, a living modified organism, a method of transforming an organism, and a method of producing a target product.