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Advances in DNA Parts Assembly for Synthetic Biology
Introduction to DNA Parts Assembly
DNA parts assembly is a cornerstone of synthetic biology, enabling the construction of complex genetic circuits and metabolic pathways. Various methods have been developed to facilitate the efficient and accurate assembly of multiple DNA parts, each with unique advantages and applications.
Unique Nucleotide Sequence (UNS)-Guided Assembly
UNS-Guided Assembly is a method designed to address the challenges posed by sequence redundancies in complex genetic constructs. This technique uses unique nucleotide sequences flanking individual DNA parts to guide their ordered assembly. It allows for the rapid and accurate construction of sequenced constructs or combinatorial libraries within a few days, without the need for specialized equipment1.
PaperClip Method for Flexible Assembly
The PaperClip Method offers a flexible approach to assembling multiple DNA parts from existing libraries. This method does not require restriction enzymes or reamplification to add end homology. Instead, it uses double-stranded oligonucleotides, or "Clips," to direct the order of assembly. This technique can efficiently assemble at least six DNA parts in any order within several hours2.
Ligase Cycling Reaction (LCR) for High-Throughput Assembly
Ligase Cycling Reaction (LCR) is a one-step, scarless DNA assembly method that uses single-stranded bridging oligos and a thermostable ligase. LCR can assemble up to 20 DNA parts into constructs as large as 20 kb with high accuracy and minimal errors. It outperforms other methods like circular polymerase extension cloning (CPEC) and Gibson isothermal assembly, making it ideal for high-throughput and automated DNA assembly3.
CasEMBLR for In Vivo Assembly and Integration
CasEMBLR leverages CRISPR/Cas9 to facilitate the in vivo assembly and chromosomal integration of DNA parts in Saccharomyces cerevisiae. This method allows for marker-free multiloci integration, enabling the construction of complex pathways and the development of cell factories. CasEMBLR has been successfully used to introduce a carotenoid pathway and create a tyrosine production strain4.
Seamless Assembly for Functional Devices
A new method for Seamless Assembly allows the integration of blunt-end double-strand nucleic acid parts into complex biological devices and multi-device systems. This technique simplifies and speeds up the optimization of device and system development by enabling combinatorial assembly without pre-assembly manipulation5.
Standards for Plant Synthetic Biology
The introduction of a Common Syntax for Type IIS-Mediated Assembly has standardized the assembly of eukaryotic transcriptional units. This standard, developed by the international plant science and synthetic biology communities, facilitates the creation of interoperable and reusable DNA parts, accelerating plant bioengineering6.
High-Throughput DNA Part Characterization
A novel technique for High-Throughput DNA Part Characterization combines combinatorial DNA part assembly, quantitative fluorescence assays, and barcode tagging-based long-read sequencing. This method allows for the rapid and accurate characterization of DNA parts, increasing part diversity and aiding in the construction of genetic circuits and metabolic pathways7.
BASIC Standard for Idempotent Cloning
The Biopart Assembly Standard for Idempotent Cloning (BASIC) uses orthogonal linker-based DNA assembly to achieve high accuracy in multi-part assemblies. This method allows for efficient parallel assembly and provides a simple, single-tier organization for maintaining parts and constructs8.
Golden Gate Assembly for Large Constructs
Golden Gate Assembly (GGA) has been optimized to construct large DNA targets from many smaller parts in a single reaction. This method successfully assembled a 40 kb T7 bacteriophage genome from 52 parts, demonstrating its potential for rapid and efficient genome construction9.
Conclusion
The advancements in DNA parts assembly methods have significantly enhanced the capabilities of synthetic biology. Techniques like UNS-guided assembly, PaperClip, LCR, CasEMBLR, and others provide researchers with powerful tools to construct complex genetic circuits and metabolic pathways efficiently and accurately. These methods are paving the way for innovative applications in bioengineering, therapeutic development, and beyond.
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Most relevant research papers on this topic
Unique nucleotide sequence–guided assembly of repetitive DNA parts for synthetic biology applications
Unique nucleotide sequence (UNS)-guided assembly enables researchers to efficiently assemble multiple DNA parts into high-quality constructs or libraries in 2-5 days, with no specialized equipment needed.
PaperClip: rapid multi-part DNA assembly from existing libraries
PaperClip allows rapid assembly of multiple DNA parts from existing libraries, enabling bioengineering and synthetic biology applications.
Rapid and reliable DNA assembly via ligase cycling reaction.
Ligase cycling reaction (LCR) enables rapid, reliable, and cost-effective assembly of up to 20 DNA parts into complex DNA constructs, outperforming existing methods in synthetic biology.
Highly CitedCasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae.
CasEMBLR enables marker-free multiloci integration of in vivo assembled DNA parts in Saccharomyces cerevisiae, improving genome engineering and cell factory development.
Highly CitedSeamless assembly of DNA parts into functional devices and higher order multi-device systems
This new method allows seamless assembly of functionally tested nucleic acid parts into complex biological devices and systems, simplifying and speeding optimization of device and system development.
Rigorous JournalStandards for plant synthetic biology: a common syntax for exchange of DNA parts.
This standard for Type IIS restriction endonuclease-mediated assembly enables facile assembly of eukaryotic transcriptional units, accelerating plant bioengineering.
Highly CitedNovel High-Throughput DNA Part Characterization Technique for Synthetic Biology
This study presents a novel high-throughput DNA part characterization technique that increases throughput by combinatorial DNA part assembly, solid plate-based quantitative fluorescence assay, and barcode tagging-based long-read sequencing, enabling rapid identification of DNA parts and their combinations in synthetic biology.
In Vitro StudyBASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology.
The BASIC standard for DNA assembly provides efficient, parallel assembly with high accuracy, enabling synthetic biology with single-tier organization.
Highly Cited