Assembly Of Multiple DNA Fragments Research Articles

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.

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.

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.

Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline.

Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one-pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline. © 2020 The Authors. Basic Protocol 1: Performing a typical Golden Gate cloning reaction Basic Protocol 2: Accommodating a vector to Golden Gate cloning Basic Protocol 3: Accommodating an insert to Golden Gate cloning Basic Protocol 4: Generating small standardized parts compatible with hierarchical modular cloning (MoClo) using level 0 vectors Alternate Protocol: Generating large standardized parts compatible with hierarchical modular cloning (MoClo) using level -1 vectors Basic Protocol 5: Assembling transcription units and multigene constructs using level 1, M, and P MoClo vectors.

A CRISPR-Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis.

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

Assembly of Complex Pathways Using Type IIs Restriction Enzymes.

Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. We describe here a protocol for assembly of multigene constructs using the modular cloning system (MoClo). Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Rapid Assembly of Yeast Expression Cassettes for Phenylpropanoid Biosynthesis in Saccharomyces cerevisiae

Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts. Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study, a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S. cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAG-CAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis gene was efficiently constructed to be the starting platform for the production of plant natural products in genetically-engineered S. cerevisiae.

Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens.

Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year.

In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.

BioPartsBuilder: a synthetic biology tool for combinatorial assembly of biological parts.

Summary: Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfies design constraints. We developed BioPartsBuilder as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. It retrieves biological sequences, enforces compliance with assembly design standards and provides a fabrication plan for each fragment.Availability and implementation: BioPartsBuilder is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38). Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder.Contact: joel.bader@jhu.eduSupplementary information: Supplementary data are available at Bioinformatics online.

AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach.

Assembly cloning is increasingly replacing conventional restriction enzyme and DNA-ligase-dependent cloning methods for reasons of efficiency and performance. Here, we describe AQUA (advanced quick assembly), a simple and versatile seamless assembly cloning approach. We demonstrate the applicability and versatility of AQUA Cloning in selected proof-of-principle applications including targeted insertion-, deletion- and site-directed point-mutagenesis, and combinatorial cloning. Furthermore, we show the one pot de novo assembly of multiple DNA fragments into a single circular plasmid encoding a complex light- and chemically-regulated Boolean A NIMPLY B logic operation. AQUA Cloning harnesses intrinsic in vivo processing of linear DNA fragments with short regions of homology of 16 to 32 bp mediated by Escherichia coli. It does not require any kits, enzymes or preparations of reagents and is the simplest assembly cloning protocol to date.

Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus

This article describes a rapid, highly efficient and versatile method for seamlessly assembling multiple DNA fragments into a vector at any desired position. The inserted fragments and vector backbone were amplified by high-fidelity PCR containing 20 bp to 50 bp overlapping regions at 3′ and/or 5′ termini. These linearised fragments were equimolarly mixed, and then cyclised in a prolonged overlap extension PCR without adding primers. The resulting PCR products were DNA multimers that could be directly transformed into host strains, yielding the desired chimeric plasmid. The proposed method was illustrated by constructing an Escherichia coli co-expression vector. The feasibility of the method in Lactobacillus was further validated by assembling an E. coli–Lactobacillus shuttle vector. Results showed that three to four fragments could be simultaneously and precisely inserted in a vector in only 2–3 days using the proposed method. The acceptable transformation efficiency was determined through the tested host strains; more than 95% of the colonies were positive transformants. Therefore, the proposed method is sufficiently competent for high-efficiency insertion of multiple DNA fragments into a plasmid and has theoretically good application potential for gene cloning and protein expression because it is simple, easy to implement, flexible and yields highly positive clones.

A tool kit for rapid cloning and expression of recombinant antibodies.

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain

Newcastle disease virus (NDV) can cause serious diseases and substantial economic losses to the poultry industry. To gain a better understanding of NDV pathogenesis, several reverse genetics systems for different NDV strains have been established. However, the construction of infectious cDNA clone by conventional restriction digestion/ligation cloning methods is a time-consuming process and has many drawbacks by its nature. To address the problems, we employed a novel and robust ligation-independent cloning (LIC) method for efficient assembly of multiple DNA fragments. Using this method, we successfully generated a NDV minigenome construct within three weeks by assembling RT-PCR products of the VG/GA strain genomic termini and a cDNA coding for the green fluorescence protein (GFP), as a reporter, into a modified pBluescript vector. Co-transfection of the NDV minigenome with three supporting plasmids expressing the N, P, and L proteins into MVA-T7 infected HEp-2 cells and followed by infection with NDV VG/GA resulted in the minigenome replication, transcription, and packaging as evidenced by the reporter gene GFP expression. These results suggest that this LIC approach is a powerful tool for all sequence-independent DNA cloning and multi-DNA fragment assembly, which has a potential application for rapid development of gene therapy and recombinant vaccines.

Phosphorothioate-based DNA recombination: an enzyme-free method for the combinatorial assembly of multiple DNA fragments.

Rational guided generation of protein chimeras has developed into an attractive approach in protein engineering for tailoring catalytic and biophysical properties of enzymes. Combinatorial recombination of structural elements or whole protein domains is still technically challenging due to sequence dependent biases diminishing the overall quality of resulting chimeric libraries. Since methods for generating such libraries are often limited by a low frequency of crossover points and suffer from challenges in handling, we developed the phosphorothioate-based DNA recombination method (PTRec). PTRec is an enzyme-free method and only requires a short stretch of four amino acids that is identical among the proteins to be recombined in order to define a single crossover point. In a PTRec-generated chimeric library that shuffled five domains of phytase using genes from three different species, 88% of 42 randomly picked and sequenced genes were efficiently recombined. Furthermore, PTRec is a technically simple, fast, and reliable method that can be used for domain- and exon-shuffling or recombination of DNA fragments in general.

In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

PHUSER (Primer Help for USER): a novel tool for USER fusion primer design

Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (Tm), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3′-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/.