Template Region Directing Site-Specific Phage Mutagenesis
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Solution Overview
Problem
Current methods for generating diversity in Bordetella bacteriophages to adapt to changing bacterial hosts are limited in their ability to efficiently switch tropism and infect specific bacteria, as they rely on complex programs of gene expression and receptor interactions that are not easily modifiable.
Innovation Solution
A nucleic acid molecule with a variable region (VR) operably linked to a template region (TR) that directs site-specific mutagenesis, allowing for directional and adenine-mediated diversification of VR sequences, enabling the phage to adapt to different host bacteria by altering ligand-receptor interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If complex programs of gene expression and receptor interactions are used for tropism switching, then the phage can adapt to different host bacteria, but the system becomes difficult to modify and control
Solution Approach 1:
The patent extracts the essential function of tropism switching from the complex gene expression program and isolates it to a specific template region (TR) that directs site-specific mutagenesis. This separates the diversification function from the complex regulatory network, allowing controlled modification of host range without manipulating the entire gene expression system.
Solution Approach 2:
The template region (TR) acts as an intermediary element that mediates between the phage genome and the host bacterium. It contains adenine residues that serve as templates for directed mutagenesis in the variable region (VR), enabling controlled adaptation to different hosts without requiring complex gene expression changes.
2Productivity
If site-specific mutagenesis is used to diversify VR sequences, then tropism switching efficiency is improved, but the system requires precise control of mutagenesis direction
Solution Approach 1:
The template region contains specific adenine residues at defined positions that locally direct mutagenesis to corresponding positions in the variable region. This local quality approach allows precise control over which nucleotides are mutated and in what direction, achieving efficient tropism switching with controllable mutagenesis.
Solution Approach 2:
The template region is pre-configured with specific adenine residues that determine the pattern and direction of mutagenesis before infection occurs. This preliminary arrangement of template adenines ensures that mutagenesis follows a predetermined path, making the directionality of viral adaptation predictable and controllable.
3Adaptability or versatility
If adenine-mediated diversification is applied to VR sequences, then host range is expanded, but the mechanism becomes more specialized and less flexible
Solution Approach 1:
The template region with its adenine residues serves multiple functions: it acts as a mutagenesis template, determines host range specificity, and enables controlled diversification of the variable region. This multi-functionality allows the specialized adenine-mediated mechanism to handle diverse host bacteria while maintaining a unified, manageable system rather than requiring separate mechanisms for each host type.
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 efficient tropism switching, enabling the phage to infect both virulent and avirulent bacteria by generating plasticity in ligand-receptor interactions through targeted mutagenesis, expanding the host range and adaptability of the phage.
Implementation Method 1
a reverse transcriptase-mediated exchange of information between two regions, one serving as a donor template region (TR) and the other as a recipient of variable sequence information
Implementation Method 2
site-directed, adenine-specific mutagenesis
Data Source
AI summary
This invention relates to the diversification of nucleic acid sequences by use of a nucleic acid molecule containing a region of sequence that acts as a template for diversification. The invention thus provides nucleic acid molecules to be diversified, as well as those which act as the template region (TR) and in concert with the TR for directional, site-specific diversification. Further provided are methods of preparing and using these nucleic acid sequences.