Assembly Of DNA Fragments Research Articles

Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments

The Golden Gate method is an efficient tool for seamless assembly of multiple DNA fragments, which uses Type IIS restriction endonucleases, cleaving the DNA outside of their recognition site to release DNA parts from PCR fragments or entry clones, thus allowing the design of overhangs for ligation at will. However, the construction of the entry clones requires the use of other restriction enzyme(s) or cloning techniques and different entry vectors for the individual overhangs. Here, we present a simplified Golden Gate cloning approach termed Golden EGG. It features (1) a single entry vector with a specific cloning site to host the DNA parts; (2) a unique primer design to create the restriction enzyme recognition site to release the fragments with the overhangs at will; (3) the use of a single Type IIS enzyme for the construction of both the entry and destination clones; (4) a specific temperature profile during the digestion-ligation reaction. Our user-friendly, streamlined method retains the key attributes of the Golden Gate technique, while offering the potential to generate compatible parts with any existing Golden Gate toolkit and to be accessible to a wide user base without the need for extensive acquisition of new vectors or expensive enzymes.

Modern Approaches to de novo Synthesis of Extended DNA Fragments: Assembly of a Wide Repertoire of Sequences.

The standardization of DNA fragment assembly methods for many tasks of synthetic biology is crucial. This is necessary for synthesizing a wider repertoire of sequences, as well as for further automation and miniaturization of such reactions. In this work, we proposed conditions for the assembly of DNA fragments from chemically synthesized oligonucleotides and we identified the errors occurring in the sequence under these conditions. Additionally, we proposed conditions for further combining synthetic fragments into larger DNA fragments. We showed that the optimized conditions are suitable for the assembly of a wide range of sequences.

Rainbow screening: Chromoproteins enable visualized molecular cloning.

Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue-white screening, have been developed for visualization. However, these methods typically require additional genetic manipulations and costs. To simplify the process of visualized molecular cloning, here we report Rainbow Screening, a method that combines Gibson Assembly with chromoproteins to distinguish Escherichia coli (E. coli) colonies by naked eyes, eliminating the need for additional genetic manipulations or costs. To illustrate the design, we select both E. coli 16s rRNA and sfGFP expression module as two inserted fragments. Using Rainbow Screening, false positive colonies can be easily distinguished on LB-agar plates. Moreover, both the assembly efficiency and the construct accuracy can exceed 80%. We anticipate that Rainbow Screening will enrich the molecular cloning methodology and expand the application of chromoproteins in biotechnology and synthetic biology.

Research Progress on the Assembly of Large DNA Fragments.

Synthetic biology, a newly and rapidly developing interdisciplinary field, has demonstrated increasing potential for extensive applications in the wide areas of biomedicine, biofuels, and novel materials. DNA assembly is a key enabling technology of synthetic biology and a central point for realizing fully synthetic artificial life. While the assembly of small DNA fragments has been successfully commercialized, the assembly of large DNA fragments remains a challenge due to their high molecular weight and susceptibility to breakage. This article provides an overview of the development and current state of DNA assembly technology, with a focus on recent advancements in the assembly of large DNA fragments in Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. In particular, the methods and challenges associated with the assembly of large DNA fragment in different hosts are highlighted. The advancements in DNA assembly have the potential to facilitate the construction of customized genomes, giving us the ability to modify cellular functions and even create artificial life. It is also contributing to our ability to understand, predict, and manipulate living organisms.

Gene synthesis design: a pythonic approach.

Researchers often need to synthesize genes of interest in this era of synthetic biology. Gene synthesis by PCR assembly of multiple DNA fragments is a quick and economical method that is widely applied. Up to now, there have been a few software solutions for designing fragments in gene synthesis. However, some of these software solutions use programming languages that are not popular now, other software products are commercial or require users to visit servers. In this study, we propose a Python program to design DNA fragments for gene synthesis. The algorithm is designed to meet the experimental needs. Also, the source code with detailed annotation is freely available for all users. Furthermore, the feasibility of the algorithm and the program is validated by experiments. Our program can be useful for the design of gene synthesis in the labs and help the study of gene structure and function.

In Vivo DNA Assembly Using the PEDA Method.

Simple and efficient DNA assembly methods have been widely used in synthetic biology. Here, we provide the protocol for the recently developed PEDA (phage enzyme-assisted in vivo DNA assembly) method for direct in vivo assembly of individual DNA parts in multiple microorganisms, such as Escherichia coli, Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. PEDA allows in vivo assembly of DNA fragments with homologous sequences as short as 5bp, and the efficiency is comparable to the prevailing in vitro DNA assembly, which will broadly boost the rapid progress of synthetic biology.

Building an Open-Source DNA Assembler Device

Golden Gate DNA assembly reactions are one of the most widely used techniques for the construction of genetic systems [1]. This technique enables the modular and combinatorial assembly of multiple standardized DNA fragments into a single piece. This is achieved through reiterative cycles of enzymatic cleavage and ligation steps that take place at 37°C and 16°C, respectively [1]. The thermal cycling required can be performed by incubating the samples in thermocyclers. However, these proprietary devices are expensive and highly demanded for Polymerase Chain Reaction (PCR), the popular technique for DNA amplification and diagnostics.

Enzymatic Assembly of DNA Nanostructures and Fragments with Sequence Overlaps.

Homologous recombination, an evolutionarily conserved DNA double-strand break repair pathway to protect genome stability, has long been exploited for the in vivo and in vitro assembly of multiple DNA duplex fragments in molecular cloning. Whether such methods can also be applied in the self-assembly of DNA nanostructures remains underexplored. Here, we report an enzymatic approach for the self-assembly of high-order DNA constructs with overlapping segments. In our system, a DNA polymerase with exonuclease activity was introduced to produce ssDNA overhangs for specific sticky end cohesion, and as many as 25 DNA structural units were designed to be hierarchically assembled. Using this approach, we successfully constructed a variety of high-order DNA nanostructures, including tubes and extended oligomers, from homogeneous assembly and custom multimers from heterogeneous assembly. Our strategy expands the construction toolbox of complex DNA nanostructures and highlights the potential to enhance the assembly of duplex fragments in molecular cloning.

Heterologous Production of Artemisinin in Physcomitrium patens by Direct in vivo Assembly of Multiple DNA Fragments.

The sesquiterpene lactone compound artemisinin is a natural medicinal product of commercial importance. This Artemisia annua-derived secondary metabolite is well known for its antimalarial activity and has been studied in several other biological assays. However, the major shortcoming in its production and commercialization is its low accumulation in the native plant. Moreover, the chemical synthesis of artemisinin is difficult and expensive due to its complex structure. Hence, an alternative and sustainable production system of artemisinin in a heterologous host is required. Previously, heterologous production of artemisinin was achieved by Agrobacterium-mediated transformation. However, this requires extensive bioengineering of modified Nicotiana plants. Recently, a technique involving direct in vivo assembly of multiple DNA fragments in the moss, P. patens, has been successfully established. We utilized this technique to engineer artemisinin biosynthetic pathway genes into the moss, and artemisinin was obtained without further modifications with high initial production. Here, we provide protocols for establishing moss culture accumulating artemisinin, including culture preparation, transformation method, and compound detection via HS-SPME, UPLC-MRM-MS, and LC-QTOF-MS. The bioengineering of moss opens up a more sustainable, cost effective, and scalable platform not only in artemisinin production but also other high-value specialized metabolites in the future.

Synthesis and assembly of full-length cyanophage A-4L genome

Artificial cyanophages are considered to be an effective biological method to control harmful cyanobacterial bloom. However, no synthetic cyanophage genome has been constructed and where its obstacles are unclear. Here, we survey a stretch of 16 kb length sequence of cyanophage A-4L that is unclonable in Escherichia coli. We test 12 predicted promoters of cyanophage A-4L which were verified all active in E. coli. Next, we screen for eight ORFs that hindered the assembly of intermediate DNA fragments in E. coli and describe that seven ORFs in the 16 kb sequence could not be separately cloned in E. coli. All of unclonable ORFs in high-copy-number plasmid were successfully cloned using low-copy-number vector, suggesting that these ORFs were copy-number-dependent. We propose a clone strategy abandoned the promotor and the start codon that could be applied for unclonable ORFs. Last, we de novo synthesized and assembled the full-length genome of cyanophage A-4L. This work deepens the understanding of synthetic cyanophages studies.

ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms

The assembly of DNA fragments is extremely important for molecular biology. Increasing numbers of studies have focused on streamlining the laborious and costly protocols via in vivo DNA assembly. However, the existing methods were mainly developed for Escherichia coli or Saccharomyces cerevisiae, whereas there are few direct in vivo DNA assembly methods for other microorganisms. The use of shuttle vectors and tedious plasmid extraction and transformation procedures make DNA cloning in other microorganisms laborious and inefficient, especially for DNA library construction. In this study, we developed a "phage enzyme-assisted in vivo DNA assembly" (PEDA) method via combinatorial expression of T5 exonuclease and T4 DNA ligase. PEDA facilitated the in vivo assembly of DNA fragments with homologous sequences as short as 5 bp, and it is applicable to multiple microorganisms, such as Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. The cloning efficiency of optimized PEDA is much higher than that of the existing in vivo DNA assembly methods and comparable to that of in vitro DNA assembly, making it extremely suitable for DNA library cloning. Collectively, PEDA will boost the application of in vivo DNA assembly in various microorganisms.

A CRISPR-Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola.

Gene editing technology plays an extremely significant role in synthetic biology and metabolic engineering. Traditional genetic manipulation methods, such as homologous recombination, however, are inefficient, time-consuming, and barely feasible when disrupting multiple genes simultaneously. Starmerella bombicola, a nonconventional yeast that overproduces sophorolipids, lacks convenient genetic tools for engineering strains. Here, we developed an efficient CRISPR-Cas9 genome editing technology by combining molecular element mining and expression system optimization for S. bombicola. This CRISPR-Cas9 system improved the efficiency of gene-integration/target gene-introducing disruption by homology-directed repair and realized the multi-gene simultaneous disruptions. Based on this CRISPR-Cas9 system, we also further constructed an engineered strain via the in vivo assembly of multiple DNA fragments (10 kb) that can produce acid-type sophorolipids. These results showed that the CRISPR-Cas9 system may be an efficient and convenient strategy to perform genetic manipulation in S. bombicola.

One-Step In Vivo Assembly of Multiple DNA Fragments and Genomic Integration in Komagataella phaffii.

The methylotrophic yeast species Komagataella phaffii (synonym: Pichia pastoris) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on K. phaffii, advanced methods such as in vivo DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step in vivo assembly of multiple DNA fragments and genomic integration in K. phaffii. To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus in vivo, a K. phaffii strain, lacking a non-homologous end joining-related protein, DNA ligase IV (Dnl4p), that has been reported to improve gene targeting efficiency by homologous recombination, was used. Using green fluorescent protein along with the lycopene biosynthesis, we showed that our method that included a Dnl4p-defective strain permits direct and easy engineering of K. phaffii strains.

Application of Restriction Free (RF) Cloning in Circular Permutation.

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

Advances in the reverse genetics system for RNA viruses

RNA viruses are responsible for several infectious diseases, including dengue fever, Zika fever, and COVID-19. Reverse genetics is a powerful tool to elucidate which domain or mutations in RNA viruses determine their pathogenicity and ability to evade antiviral drugs and host immune response. Previous reverse genetics systems for flaviviruses and coronaviruses have been technically challenging and time-consuming, thereby hampering the further understanding of events during viral evolution. A novel reverse genetics system-circular polymerase extension reaction (CPER)-has been developed to overcome this limitation. CPER is based on PCR-mediated assembly of DNA fragments that encode the whole genome of these viruses. CPER requires a relatively short time to introduce specific mutations into the viral genome of flaviviruses and SARS-CoV-2. In this review article, we explain the mode of action of this system and discuss the future direction of reverse genetics for RNA viruses.

Efficient de novo assembly and modification of large DNA fragments.

Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes. The construction of the large, gigabase (Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly. But for the synthesis and assembly of such large-scale genomes, the development of new or expanded methods is required. In this study, we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases (kb) or above from scratches and describe an efficient method for "scar-free" engineering of the assembled sequences. Our method, therefore, should provide a standard framework for producing long DNA molecules, which are critical materials for synthetic genomics and metabolic engineering.

Construction of Sedoheptulose-1,7-Bisphosphatase (Sbpase) for Manipulation in Guard Cells of Arabidopsis thaliana L.

Guard cells control the stomata through which exchange of gas takes place in plants by balancing between CO2 uptake for photosynthesis and water loss through transpiration leading to ultimate plant water use efficiency (WUE). Due to climate change, sustainable agriculture will therefore require a major reduction in plant water use hence stomata have become potential target for manipulation. Understanding the signal mechanisms of stomata is therefore critically important in facilitating an understanding of stomatal regulation. The use of molecular tools and techniques to manipulate chloroplast metabolism specifically in the guard cells are needed to elucidate signals associated with stomatal behaviour towards crop improvement. Ability to assemble multiple or complex DNA molecules containing large number of genetic elements is an essential part of genetic engineering and in order to understand the involvement of guard cell photosynthesis in stomatal function, genetic manipulation of photosynthetic enzymes specifically in guard cells is necessary. This study employed the manipulation and construction of the enzyme Sedoheptulose-1,7-Bisphosphatase (SBpase) by using the golden gate cloning technique and the bioinformatics system- geneious. Constructs were designed to alter expression of the SBPase gene in a cell specific manner driven by the guard cell promoter KST1 in the model plant Arabidopsis thaliana L. The construct design for the sense plasmid vectors allowed efficient assembly of multiple DNA fragments in a single reaction based on the type IIs restriction enzyme. The potentials of manipulating guard cell specific metabolism are therefore enormous and the increase or decrease of photosynthetic genes could be assessed and their impacts on plant development documented accordingly.

Creation of Golden Gate constructs for gene doctoring

BackgroundGene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps.ResultsWe constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker.ConclusionsOur plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.