VipR Regulator and PvipR Promoter for Stationary-Phase Vip3A Expression
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Solution Overview
Problem
The upstream region of the vip3A gene in Bacillus thuringiensis strain kurstaki HD73 is insufficient for inducing gene expression, and a specific regulator, VipR, is required for its transcription, which is not adequately controlled by sporulation-specific sigma factors.
Innovation Solution
An expression system utilizing the VipR regulator and its promoter, PvipR, is developed to regulate the expression of genes, including vip3A, in Bacillus strains, using a sequence upstream of the Sigma A-specific −10 box to direct transcription during the stationary phase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the upstream region of vip3A is used to induce gene expression, then gene expression should be activated, but expression is not induced without the VipR regulator
Solution Approach 1:
The patent introduces the VipR regulator as an intermediary component that mediates between the promoter region and the vip3A gene. The expression system includes both the upstream region of vip3A and the vipR regulator gene, where VipR acts as a transcriptional activator that binds to the promoter to enable reliable gene expression induction.
2Duration of action of moving object
If sporulation-specific sigma factors control cry gene expression, then expression occurs during sporulation, but vip3A expression is not adequately controlled and remains low
Solution Approach 1:
The patent changes the regulatory parameter from sporulation-specific sigma factor control to VipR regulator control. The expression system is designed to be controlled by VipR, which is active during stationary phase rather than sporulation, thereby changing the temporal and regulatory parameters of gene expression to achieve both proper timing and high productivity.
3Productivity
If the expression system uses the VipR regulator and PvipR promoter, then high-level expression is achieved during stationary phase, but the system complexity increases
Solution Approach 1:
The expression system utilizes the bacterium's own stationary phase physiology to trigger VipR activation and subsequent gene expression. The system is self-regulating through the natural transition to stationary phase, eliminating the need for external induction mechanisms or complex regulatory circuits, thereby achieving high productivity without excessive complexity.
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
The system enables high-level and sustained expression of proteins of interest, such as Vip3A, during the stationary phase, enhancing insecticidal activity against lepidopteran insects and bypassing resistance to Cry toxins.
Implementation Method 1
the expression of Vip3A in the HD73 strain containing pBMB299 is controlled by a regulator whose gene is located on this same plasmid. This regulator gene called vipR is responsible for vip3A gene transcription during the stationary phase.
Data Source
AI summary
The present invention relates to a novel regulator VipR able to regulate the expression of genes, and more specifically the expression of vip3A. It also relates to an expression system allowing the expression of a protein of interest through the VipR regulation. This expression system can be used to transform Bacillus strains.