Bacillus Subtilis Virus Vector Plasmid Production With Low Endotoxin
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Solution Overview
Problem
Existing methods for producing virus vector plasmids are limited to Escherichia coli and do not utilize Bacillus subtilis, which has potential advantages for plasmid production.
Innovation Solution
A method for producing virus vector plasmids in Bacillus subtilis by introducing a nucleic acid sequence for virus vector production into a host cell, amplifying the plasmid under specific conditions, and utilizing Bacillus subtilis as a host for replication and amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If virus vector plasmid production is performed in Escherichia coli, then production capability is established, but endotoxin contamination occurs
Solution Approach 1:
The invention changes the host cell parameter from Escherichia coli to Bacillus subtilis, fundamentally altering the biological system to eliminate endotoxin production while maintaining plasmid replication capability through species-appropriate optimization
Solution Approach 2:
The invention uses Bacillus subtilis as a disposable host system that can be easily cultured and processed, where the host cells are grown, harvested for plasmid production, and then discarded, eliminating the need for complex endotoxin removal procedures
2Object-generated harmful factors
If virus vector plasmid production is performed in Bacillus subtilis, then endotoxin levels are reduced, but production method must be developed
Solution Approach 1:
The invention optimizes multiple parameters including replication origin selection, promoter strength, cultivation temperature, and harvest timing to achieve high-yield plasmid production in Bacillus subtilis without complex procedures
Solution Approach 2:
The invention utilizes the natural transformation capability and cell-free DNA release properties of Bacillus subtilis to automatically achieve high plasmid yields without requiring complex induction or harvesting procedures
3Productivity
If plasmid amplification is performed under standard conditions, then amplification efficiency is maintained, but CCC purity decreases
Solution Approach 1:
The invention employs periodic sampling during the amplification process to monitor plasmid quality and determine optimal harvest timing, ensuring high CCC purity while maintaining efficient production
Solution Approach 2:
The invention replaces complex mechanical purification systems with optimized biological amplification conditions in Bacillus subtilis that naturally favor high CCC purity through controlled replication and harvest parameters
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 allows for the production of virus vector plasmids with reduced endotoxin levels and high CCC purity, enabling efficient virus vector production in Bacillus subtilis.
Implementation Method 1
placing a host cell comprising the plasmid under a condition where the plasmid is amplified
Implementation Method 2
the cell-free DNA release upon completion of the amplification
Data Source
AI summary
The present disclosure pertains to production of a virus vector plasmid in Bacillus subtilis. According to one aspect, the present disclosure provides a method for producing a virus vector plasmid having a sequence to be replicated in Bacillus subtilis. The method includes a step for forming a plasmid in a host cell by introducing, into the host cell, a nucleic acid that has a sequence to be replicated in Bacillus subtilis and that includes a nucleic acid sequence for producing a virus vector. In one embodiment, Bacillus subtilis could have the ability to form a plasmid from a nucleic acid acquired from outside, and therefore, in this method, the nucleic acid introduced does not have be a plasmid.