Gene Stacking Vectors for 300 kb DNA Assembly and Editing
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Solution Overview
Problem
Existing vector systems in synthetic biology for plants have a cargo size limit of about 25 kilobases, limiting the assembly of multi-gene pathways and circuits, and lack flexibility for post-assembly modifications.
Innovation Solution
A gene stacking system with engineered bacterial cells, including recombinase-expressing E. coli strains, and vectors capable of handling up to 300 kb of target DNA inserts, enabling recombineering for editing and integration of multiple DNA inserts using PhiC31 and FLP recombinases, compatible with Type IIS restriction enzyme-based cloning technologies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing vector systems (e.g., GoldenBraid) are used for assembling multi-gene pathways, then the assembly process is manageable, but the cargo size is limited to about 25 kb which prevents assembly of larger multi-gene circuits
Solution Approach 1:
The patent divides the large multi-gene construct into multiple smaller donor vectors, each containing a portion of the target DNA insert. These segmented donor vectors (e.g., Donor 1, Donor 2, etc.) are sequentially integrated into a single acceptor vector through multiple rounds of recombination, ultimately achieving assembly of large constructs exceeding 25 kb that would be impossible with a single vector system
Solution Approach 2:
The patent implements a nested structure where multiple donor vectors are sequentially integrated into an acceptor vector. Each donor vector contains DNA inserts that are nested within the growing construct in the acceptor vector, similar to nested dolls. This allows progressive building of large multi-gene circuits by nesting additional genetic elements into the acceptor vector across multiple integration rounds
2Adaptability or versatility
If large multi-gene constructs are built using existing systems, then the pathways can be assembled, but modification after assembly is impossible requiring researchers to start from entry-level DNA parts
Solution Approach 1:
The patent creates a dynamic system where the acceptor vector maintains flexibility for post-assembly modification through retained recombination sites (attP/attB sites) and compatibility with recombineering methods. This allows the construct to be dynamically modified after assembly by introducing new donor vectors or using homology-directed recombination, eliminating the need to dismantle and rebuild entire constructs
Solution Approach 2:
The patent introduces recombineering as an intermediary method that enables modification of large constructs without requiring complete deconstruction. By using homologous recombination with donor DNA fragments containing desired modifications, researchers can directly edit the assembled construct through an intermediary recombination process rather than starting from entry-level parts
3Length of stationary object
If multiple donor vectors are used for gene stacking, then larger DNA inserts up to 300 kb can be contained, but the system complexity increases with multiple integration rounds
Solution Approach 1:
The patent creates universal donor vectors and acceptor vectors with standardized recombination sites (attP/attB) that can be used across multiple integration rounds. The same vector architecture and recombination system serve all integration steps, providing multi-functionality that simplifies the process despite multiple rounds being required. This universal system allows consistent handling of each donor vector integration using the same molecular biology tools and conditions
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 assembly and flexible editing of large DNA constructs, overcoming the size limitations of existing systems and allowing for seamless integration and modification of genetic circuits.
Implementation Method 1
enabling recombineering for editing and integration of multiple DNA inserts using PhiC31 and FLP recombinases
Implementation Method 2
the recombincering system comprises heat-shock-inducible lambda Red proteins
Data Source
AI summary
The present disclosure provides materials and methods related to gene stacking. In particular, the present disclosure provides gene stacking systems and components thereof and methods for delivering target DNA inserts to acceptor vectors and target cells such as plant cells. The provided gene stacking systems and components thereof enable an increased DNA cargo size limit, such as an acceptor vector configured to contain up to 300 kb of target DNA inserts. The provided gene stacking systems and components thereof also enable editing of constructs post-assembly. Additionally, the provided gene stacking systems are compatible with all major Type IIS cloning technologies in plants.