mv4 attP Recombination Site for Host-Independent DNA Integration
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Solution Overview
Problem
Existing site-specific recombination systems, such as those using lambda-like integrases, are limited by the need for species-specific host factors and the difficulty in modifying DNA sites, restricting their application in genome engineering across different bacterial species.
Innovation Solution
Adapting the attP site of the bacteriophage mv4 to integrate DNA into bacterial hosts using a redefined core-attP region, allowing recombination without requiring host factors, and utilizing the mv4Int integrase to integrate DNA into various bacterial genomes, including both Gram-positive and Gram-negative bacteria.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If lambda-like integrases are used for site-specific recombination, then recombination efficiency is improved, but host factor dependency increases and application scope is limited
Solution Approach 1:
The mv4 integrase system is designed to function universally across different bacterial species without requiring species-specific host factors. The integrase enzyme recognizes and binds to the engineered attP site through its specific DNA binding domain, enabling the same recombination machinery to work in both Gram-negative and Gram-positive bacteria, thus achieving multi-functionality across diverse hosts
2Reliability
If traditional attP sites are used for recombination, then integration specificity is improved, but DNA site modifiability decreases
Solution Approach 1:
The attP site is segmented into distinct functional domains: a core region containing the essential 7-bp overlap sequence that ensures specific recombination, and flanking regions that can be freely modified. This segmentation allows the core specificity-determining elements to remain intact while the surrounding sequences are engineered for different applications, such as inserting various polynucleotide sequences of interest
Solution Approach 2:
Different regions of the attP site are assigned different functional qualities: the central overlap region maintains strict sequence identity for high-fidelity recombination, while the adjacent B and B' regions allow sequence variation and engineering. This local differentiation enables simultaneous maintenance of specificity and flexibility in DNA site design
3Adaptability or versatility
If site-specific recombination systems are engineered for flexibility, then adaptability to different bacteria is improved, but system complexity increases
Solution Approach 1:
The invention extracts and isolates the essential recombination function into a self-contained attP site design that does not require extraction or addition of host factors. The mv4 integrase operates autonomously by recognizing the engineered attP site through its intrinsic DNA binding capability, eliminating the need for complex host factor systems and reducing overall system complexity while maintaining broad adaptability
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
Enables efficient and flexible integration of foreign DNA into bacterial genomes without genetic manipulation of the host, facilitating genome engineering across diverse bacterial species.
Implementation Method 1
site-specific recombination involves site-specific recombinases (SSR) that promote DNA rearrangements between two specific DNA target sites
Implementation Method 2
This region is made of two imperfect inverted repeats, where integrases monomers bind, that flanks an 'overlap' region where DNA breakage and religation occur
Data Source
AI summary
The present disclosure relates to a method for preparing a site-specific recombination polynucleotide molecule derived from the attP site of the bacteriophage mv4 and to a kit for such site-specific recombination. The kit can be used to transform procaryote hosts to integrate any polynucleotide sequence of interest.