Plasmid-Based CTX Phage Replication System for Vibrio Cholerae
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Solution Overview
Problem
Current methods for replicating CTX phage are limited by biotype-specific constraints, particularly in El Tor strains, and lack efficient systems for producing cholera toxin under laboratory conditions.
Innovation Solution
A recombinant plasmid-based CTX phage replication system is developed, incorporating the genomic sequence of CTX phage with a selection marker gene, allowing for replication in various host cells and improved infection efficiency, and a V. cholerae variant strain with a toxT protein mutation for enhanced cholera toxin production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional CTX phage replication is performed using El Tor biotype V. cholerae with previously lysogenized CTX phage, then CTX phage can be replicated and transduced, but the system is limited by biotype-specific constraints and requires CTX-CTX repeat or CTX-RS1 array
Solution Approach 1:
The invention creates a universal replication system that works across different V. cholerae biotypes (Classical, El Tor, and atypical El Tor) by using a plasmid-based CTX phage replication system that does not depend on biotype-specific constraints or pre-existing CTX arrays in the host chromosome
Solution Approach 2:
The invention introduces a plasmid as an intermediary carrier that contains the CTX phage genomic sequence and essential replication genes (rstR, rstA, rstB, ctxA, ctxB). This plasmid acts as a mediator that enables CTX phage replication in host strains that would otherwise be incompatible, eliminating the need for biotype-specific conditions
2Adaptability or versatility
If CTXcla phage is used without CTX-CTX array, then the phage cannot replicate due to lack of transducible E1 Tor strain, but the invention enables replication under laboratory conditions
Solution Approach 1:
The invention performs preliminary action by constructing a plasmid that already contains all necessary elements for CTX phage replication (genomic sequence, replication genes rstR/rstA/rstB, toxin genes ctxA/ctxB) before introducing it to the host. This pre-assembled plasmid eliminates the need for the phage to find a compatible host with pre-existing CTX arrays
Solution Approach 2:
The plasmid serves as an intermediary that provides the missing replication functionality to CTXcla phage. The plasmid contains the essential rst genes that enable replication in the absence of chromosomal CTX arrays, acting as a bridge that allows CTXcla to replicate in laboratory conditions
3Productivity
If selection marker gene is substituted for ctxA and ctxB genes in CTX phage genomic sequence, then the plasmid becomes replicable in host cells, but cholera toxin production is reduced
Solution Approach 1:
The invention applies local quality by making the plasmid copy carry a selection marker substitution (affecting local toxin production) while the host chromosome retains the wild-type ctxA and ctxB genes (maintaining overall toxin production capability). This localized modification allows replication selection without permanently compromising toxin production
Solution Approach 2:
The invention uses copying by creating a plasmid copy of the CTX phage genome that contains the selection marker substitution. This plasmid copy serves as a replicative form that can be selected and maintained, while the host chromosome preserves the original toxin genes for functional toxin production
Data Source
AI summary
The present invention relates to a plasmid-based CTX phage replication system and Vibrio cholerae strain that can be infected by CTX phage and can be used for cholera toxin production. More particularly, the present invention provides a Vibrio cholera variant strain, which expresses a toxT protein in which tyrosine at position 139 is substituted by phenylalanine through the point mutation of a toxT gene using a plasmid-based CTX phage replication system, and is used as a receptor strain which can improve CTX phage infection efficiency and allows a plurality of CTX prophages to simultaneously infect the strain and to be inserted into the chromosome thereof, which the consequent provision of the effect of increasing the production yield of a cholera toxin.