Nucleic Acid Assembly via Type IIs Restriction and Barcode Retrieval
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Solution Overview
Problem
The high cost and time-consuming nature of DNA assembly, particularly for large constructs, and the error-prone process of assembling genetic designs limit the rate at which genetic designs can be constructed and optimized in synthetic biology.
Innovation Solution
A method for high-throughput assembly and sequence verification of nucleic acid constructs using a set of nucleic acid elements with 3′ and/or 5′ flanking sequences and Type IIs restriction sites, enabling efficient assembly and retrieval of sequence-verified genetic designs through next-generation sequencing and barcode-based retrieval techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional restriction digest/ligation methods are used to assemble BioBrickTM concatemers, then modular DNA assembly is enabled, but the cost and turnaround time remain comparatively high
Solution Approach 1:
The patent segments the DNA assembly process into standardized modules with defined overhang sequences. Each BioBrick module contains specific 5' and 3' overhangs that dictate assembly order, enabling systematic construction of large concatemers through repeated modular units rather than traditional pairwise ligation
Solution Approach 2:
The patent performs preliminary actions by pre-defining overhang sequences on each BioBrick module before assembly. The 5' and 3' overhangs are designed in advance to complement specific downstream and upstream modules, respectively, allowing rapid assembly without time-consuming primer design or site selection during the assembly process
Solution Approach 3:
The patent changes the parameters of DNA assembly by using standardized overhang sequences (e.g., 4-6 base pairs) instead of traditional restriction sites. This parameter change enables high-throughput assembly by allowing multiple modules to be assembled simultaneously with defined connectivity rules, reducing both cost and turnaround time
2Length of stationary object
If traditional DNA assembly methods are used for large constructs (1000+ base pairs), then assembly is possible, but the process becomes error prone
Solution Approach 1:
The patent divides large constructs into smaller modular BioBrick units with standardized overhangs. Each module is independently verified and contains specific overhang sequences that ensure correct assembly order and orientation, reducing errors that accumulate in traditional large-construct assembly methods
Solution Approach 2:
The patent incorporates feedback mechanisms through the overhang sequence design, where each module's 5' and 3' overhangs provide directional information that ensures correct assembly. This built-in feedback system prevents misassembly by allowing only compatible modules to ligate together in the correct orientation
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 significantly reduces the time and cost of constructing and optimizing genetic designs, allowing for rapid iterative searches of genetic design spaces and enabling the assembly of large, complex genetic systems with high fidelity.
Implementation Method 1
the one or more restriction sites comprise at least one Type IIs restriction site. In some embodiments, cleavage of the Type IIs restriction site results in cohesive end overhangs at the 5′ and 3′ ends of the nucleic acid sequences such that the sequences are assembled into a continuous DNA fragment with the designed order and orientation of its components
Data Source
AI summary
Disclosed is a method of generating a set of sequence-verified nucleic acid elements for the combinatorial construction of genetic elements. The method includes: providing a plurality of nucleic acid parts; assembling nucleic acid parts to form a one or more nucleic acid elements, wherein the nucleic acid elements include at least two sequences selected from the plurality of parts; and determining the sequence of the nucleic acid elements. Further disclosed is a pool of higher-order nucleic acid constructs or amplification products thereof, comprising one or more nucleic acid elements as well as kits including a pool of sequence-verified nucleic acid elements of claims and/or a pool of higher-order nucleic acid constructs; and a plurality of primers for retrieving one or more sequence-verified nucleic acid elements and/or higher-order nucleic acid constructs.