Modular DNA Cassette Assembly via Type IIs Enzymes
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Solution Overview
Problem
Current methods for assembling complex DNA molecules are cumbersome, costly, and inefficient, particularly for large constructs, due to limitations in existing cloning technologies such as type II restriction enzymes and recombinase-based cloning, which struggle with seamless assembly and scalability.
Innovation Solution
A hybrid in vitro/in vivo method using standardized bio-elements and Golden Gate assembly with specialized entry vectors allows for efficient construction of modular DNA cassettes, enabling the assembly of multiple fragments into functional expression cassettes with seamless integration at a target locus, utilizing type IIs restriction enzymes and homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If type II restriction enzymes and ligase are used for DNA construction, then versatility in cloning strategies is achieved, but the process becomes slow and tedious with limited construct size
Solution Approach 1:
The patent divides complex DNA constructs into modular standardized expression cassettes that can be independently assembled. Each cassette contains specific functional elements (promoters, ORFs, terminators) that can be combined systematically, transforming a single complex cloning operation into multiple simpler modular assembly steps, thereby increasing productivity while maintaining versatility
Solution Approach 2:
The patent changes the parameters of the cloning system by introducing standardized connector sequences with specific overhangs and using a defined set of type IIS restriction enzymes. This standardization allows for rapid, predictable assembly of multiple cassettes without requiring new cloning strategies for each construct, thus increasing assembly speed while preserving adaptability
2Manufacturing precision
If recombinase-based cloning is used, then seamless assembly is achieved, but recombination sites remain in the final construct preventing further seamless assembly
Solution Approach 1:
The patent extracts and removes the recombinase recognition sites from the final construct through careful design of the assembly process. Type IIS restriction enzymes cut outside their recognition sites, allowing the recognition sites to be excluded from the final product while maintaining seamless joins between cassettes, thus preserving both seamlessness and future assembly capability
Solution Approach 2:
The patent uses standardized connector sequences as intermediaries between expression cassettes. These connectors contain the necessary overhangs for restriction enzyme recognition and ligation but are designed to be excluded from the final functional construct, serving as temporary mediators that enable seamless assembly without leaving residual sites in the final product
3Adaptability or versatility
If gene-synthesis and ligation are used for DNA cassette production, then custom constructs can be created, but the process becomes costly and must be repeated for each construct
Solution Approach 1:
The patent performs preliminary assembly of standardized expression cassettes using modular components with standardized connectors. Once assembled, these cassettes can be stored and reused in multiple different construct configurations, eliminating the need to repeat synthesis and ligation for each new construct, thus reducing time loss while maintaining custom construct capability
Solution Approach 2:
The patent creates universal standardized expression cassettes that can function in multiple different construct contexts. Each cassette is designed with standardized connectors that allow it to be combined with various other cassettes in different arrangements, making a single cassette design applicable to numerous different final constructs, thereby reducing repetitive work
4Adaptability or versatility
If sequential in vitro steps or reiterative recombination are used for library assembly, then complex multigene pathways can be constructed, but the process becomes rather complex
Solution Approach 1:
The patent merges multiple assembly operations into a single in vitro recombination reaction. By designing cassettes with compatible standardized connectors, multiple cassettes can be assembled simultaneously in one reaction mixture without requiring sequential steps or iterative recombination cycles, thus reducing process complexity while maintaining the ability to construct complex multigene pathways
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 enables rapid, cost-effective, and scalable assembly of complex DNA constructs, facilitating the reuse of standardized modules and reducing the complexity of DNA assembly, thereby accelerating the design and construction of organisms with novel phenotypes in synthetic biology.
Implementation Method 1
each element sequence being flanked on both sides by a type IIs restriction endonuclease cleavage site followed by the recognition site
Implementation Method 2
assembling the two or more sets of element sequences as functional expression cassettes in the at least two backbone entry vectors, using a method based on the use of restriction enzyme digestion and ligation
Implementation Method 3
the RF and LF sequences on any backbone entry vector are selected so that they can assemble by recombination in vivo with a LF or RF connector sequence
Data Source
AI summary
The present invention relates to a method based on the use of restriction enzyme digestion and ligation via the cleavage sites, thereby to prepare two or more standardized expression cassettes.