DNA Construct Balancing Anticancer and Reporter Protein Expression
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Solution Overview
Problem
Current cancer therapies face challenges in selectively targeting cancer cells while minimizing harm to normal cells, particularly due to unbalanced protein expression levels from existing inducible promoters, which limits their clinical applicability.
Innovation Solution
A DNA construct with a regulatory protein that induces both a first and second promoter, allowing balanced expression of anticancer and reporter proteins, enabling simultaneous diagnosis and treatment by controlling protein expression levels with a single regulatory element.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a Ptet promoter system is used for inducible expression, then clinical applicability is improved through doxycycline induction, but protein expression levels become unbalanced with a difference of 100:1 or more between TetA and TetR promoters
Solution Approach 1:
The patent modifies promoter sequences to adjust their expression levels. Specifically, it creates mutant versions of the Ptet promoter with altered -35 and/or -10 region sequences to reduce the expression difference between TetA and TetR promoters from 100:1 to a more balanced ratio, enabling simultaneous diagnosis and treatment protein expression
Solution Approach 2:
The patent introduces a regulatory protein system where TetR repressor binds to tetO operators to control both TetA and TetR promoter activities. This intermediary regulatory mechanism allows coordinated induction of diagnostic and therapeutic proteins through doxycycline, resolving the expression imbalance problem
2Reliability
If L-arabinose is used as an inducer for PBAD promoter, then inducible expression is achieved, but clinical application is limited because L-arabinose is not permitted for human use
Solution Approach 1:
The patent replaces the PBAD promoter system with a Ptet promoter system that responds to doxycycline. This parameter change in the inducer molecule from L-arabinose to doxycycline maintains reliable inducible expression control while enabling clinical applicability since doxycycline is approved for human use
3Measurement precision
If bacteria are administered to target cancer tissue, then specific cancer targeting is achieved, but therapeutic proteins must be expressed after clearance in reticuloendothelial systems, requiring delayed expression control
Solution Approach 1:
The patent implements a dual-promoter system where both the diagnostic reporter protein and therapeutic protein are under the control of inducible Ptet promoters. This preliminary setup allows both proteins to be expressed simultaneously upon doxycycline induction, eliminating the time delay between bacterial clearance and therapeutic protein expression while maintaining cancer-specific targeting
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 precise and balanced expression of therapeutic proteins, enhancing the effectiveness of cancer diagnosis and treatment while minimizing side effects on normal cells.
Implementation Method 1
A DNA construct with a regulatory protein that induces both a first and second promoter, allowing balanced expression of anticancer and reporter proteins
Data Source
AI summary
The present disclosure relates to a DNA construct, and a strain into which a recombinant vector comprising the DNA construct has been introduced. The DNA construct according to the present disclosure allows the expression levels of genes, operably linked downstream of first and second promoters, in a host strain or cell, to be balanced, so that cancer diagnosis and treatment may be performed simultaneously. In addition, the DNA construct of the present disclosure completely does not allow the anticancer protein and the reporter protein to be expressed at all in the absence of doxycycline, and thus it allows the anticancer protein to be expressed at an appropriate dose for cancer treatment by controlling whether or not treatment with doxycycline is performed, and at the same time, enables the size of the cancer to be monitored in real time depending on the expression level of the reporter protein.