Target Protein Production Through Antibiotic-Free Vector Maintenance
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Solution Overview
Problem
Existing methods for producing target proteins using vectors in host cells involve the risk of contamination by antibiotic resistance genes and environmental leakage, and require genetic manipulation or introduction of non-target sequences.
Innovation Solution
A method for producing target proteins using vectors that do not contain antibiotic resistance genes, recombinase recognition sequences, or genes essential for cell survival, allowing stable vector maintenance without special genetic manipulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If an antibiotic resistance gene is introduced into the vector to stably maintain it in host cells, then the vector stability is improved, but the risk of contamination of the resulting enzyme product with the antibiotic resistance gene increases
Solution Approach 1:
The invention extracts and removes the antibiotic resistance gene from the vector system entirely. Instead of using traditional antibiotic resistance genes for selection and maintenance, the patent employs a different mechanism (essential gene complementation) that does not involve antibiotic resistance markers, thereby eliminating the contamination risk while maintaining vector stability
2Stability of the object's composition
If an antibiotic resistance gene is used for vector maintenance, then the vector can be stably maintained in host cells, but the antibiotic resistance gene or antibiotic may leak into the environment causing resistant bacteria development
Solution Approach 1:
The invention removes the antibiotic resistance gene and antibiotic selection system from the vector. The patent uses an alternative mechanism where the vector contains an essential gene that complements a knockout in the host genome, allowing selection without antibiotic resistance markers and preventing environmental leakage of antibiotics and resistance genes
3Stability of the object's composition
If essential genes are knocked out in host cells to maintain the vector, then the vector can be stably maintained, but the host cell genetic structure is permanently altered
Solution Approach 1:
The invention performs preliminary genetic manipulation by knocking out the essential gene in the host cell before introducing the vector. This preliminary action creates a host cell that is dependent on the vector for survival, ensuring stable vector maintenance. The vector contains the essential gene sequence that complements the knockout, allowing the host cell to survive only when the vector is present
4Object-affected harmful factors
If recombinase recognition sequences are introduced into the vector to enable selective removal of antibiotic resistance genes, then the antibiotic resistance gene can be removed after culture, but the vector contains additional non-target sequences
Solution Approach 1:
The invention extracts and removes both the antibiotic resistance gene and the recombinase recognition sequences from the vector. The patent uses a cleaner approach where the vector simply contains the essential gene sequence without any additional sequences for gene removal mechanisms, thereby reducing vector structure complexity while still eliminating contamination risks
Data Source
AI summary
Provided is a method which can produce a target protein while stably maintaining a vector without any special genetic manipulation of host cells and without use of a drug resistance gene or the like. A method for producing a target protein including culturing cells transformed with a vector, the vector containing a gene of the target protein and not containing an antibiotic resistance gene, a recombinase recognition sequence, or a gene essential for cell survival.