T7 Expression System Plasmid Stabilization and Antibiotic-Free Selection
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Solution Overview
Problem
T7 expression systems for recombinant protein production in E. coli have low plasmid stability and rely on antibiotic resistance genes, which are undesirable for therapeutic protein production due to regulatory and safety concerns.
Innovation Solution
A T7 expression system with a prokaryotic cell containing a nucleotide construct encoding T7 RNA polymerase under an inducible promoter, integrated into the genome using site-specific recombination, and an expression vector with a plasmid stabilization system, such as the cer sequence, to enhance stability and eliminate antibiotic markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a T7 expression system with genomic anchoring of T7 polymerase is used, then the expression system is functional, but plasmid stability is low when target genes are expressed
Solution Approach 1:
The invention separates the T7 polymerase provision from the expression vector by using genomic anchoring of T7 polymerase under an inducible promoter (segmenting the system into host-integrated components and plasmid-based expression components). This segmentation allows independent optimization of plasmid stability and expression capability.
Solution Approach 2:
The invention introduces an inducible promoter as an intermediary control element between the host genome and the T7 polymerase expression. This intermediary allows conditional activation of T7 polymerase only when needed, reducing metabolic burden and improving plasmid stability while maintaining high expression capability when induced.
2Ease of manufacture
If antibiotic resistance genes are used in expression vectors, then selection and cloning are enabled, but the process is unsuitable for therapeutic protein production due to safety concerns
Solution Approach 1:
The invention extracts and removes the antibiotic resistance gene from the expression vector, eliminating the harmful element while retaining the essential cloning and selection functionality through alternative means (such as antibiotic-free selection markers or physiological selection).
Solution Approach 2:
The invention changes the selection parameter from antibiotic-based selection to alternative selection methods that do not involve antibiotic resistance genes, thereby maintaining the ease of manufacture for cloning and selection while eliminating safety concerns for therapeutic protein production.
3Reliability
If lambda phage DNA is integrated into E. coli genome for T7 polymerase expression, then T7 expression system is established, but the system contains undesirable phage DNA for pharmaceutical protein expression
Solution Approach 1:
The invention extracts only the necessary T7 polymerase gene from the lambda phage genome and integrates it into the E. coli chromosome under an inducible promoter, while leaving out the undesirable lambda phage DNA sequences. This selective extraction eliminates contamination risks while maintaining T7 expression system functionality.
Solution Approach 2:
The invention segments the lambda phage genome to isolate only the T7 polymerase gene, separating it from the rest of the phage DNA. This segmentation allows the T7 polymerase to be expressed from a minimal, safe integration that does not include harmful phage elements.
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 achieves higher plasmid stability and allows for antibiotic-free production of recombinant proteins, improving expression performance and safety, particularly in complex media.
Implementation Method 1
The T7 phage polymerase recognizes the T7 phage promoter
Implementation Method 2
nucleotide construct encoding a T7 RNA polymerase under the control of a T7 promoter and under the control of an inducible promoter
Implementation Method 3
the T7 RNA polymerase under the control of a T7 promoter and under the control of an inducible promoter by means of a 'site specific' recombination into the genome was specifically integrated into the prokaryotic cell
Data Source
Figure 1a~1b
Figure 2
Figure 3
AI summary
The invention relates to an expression system comprising a prokaryotic cell which contains a polynucleotide construct that encodes a T7 RNA polymerase under the control of a T7 promoter and under the control of an inducible promoter. The invention also relates to an expression vector which contains a gene that encodes a protein to be expressed under the control of a T7 promoter, characterized in that said expression vector comprises a plasmid stabilization system.