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Watermelon mosaic virus (WMV), a member of the genus Potyvirus, causes serious economic losses in cucurbit crops. For molecular biological studies of viruses, it is necessary to construct an infectious clone that can facilitate gene functional analysis. In this study, we constructed an infectious cDNA clone of WMV genomic RNA by Gibson assembly and evaluated its virulence and symptoms on a variety of host plants. A WMV isolate (WMV-IS-me2) collected from melon in Iksan, Jeonbuk Province, Korea in 2015 caused mosaic symptoms on leaves. Four overlapping PCR fragments of the full-length genome of this isolate and the pJL89 binary vector with overhanging ends of 40 bp were amplified by reverse transcription PCR and joined in a single isothermal reaction. The complete nucleotide sequence of the infectious cDNA clone of WMV was determined and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101. Following agro-infiltration, an infectious clone (pWMV-M, KACC95145P) systemically induced mosaic symptoms on watermelon (Citrullus lanatus), zucchini (Cucurbita pepo), melon (Cucumis melo), and Nicotiana benthamiana plants. This infectious clone may be useful for screening cucurbit varieties resistant to WMV, as well as for studying viral gene function.

Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes. [ABSTRACT FROM AUTHOR]

For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.

Both human and non-human simian adenoviruses (HAdVs and SAdVs, respectively) have been used as gene therapy and vaccine vectors. The high prevalence of HAdVs and the neutralizing antibodies associated with prior infection, may limit HAdV-based vector use in human subjects. To overcome this drawback, a vector derived from a newly isolated and characterized macaque adenovirus was constructed. SAdVs (33.9%) were screened from 115 SAdV fecal samples collected at a zoological park. One novel SAdV was isolated and the whole genome was sequenced and analyzed. The pre-existing neutralizing antibody levels were very low against this isolate (10%). Interestingly, SAdV vector constructs that lack E3 region could not produce infectious progeny in HEK293 cells, suggesting that the E3 region is necessary for SAdV replication. The absence of E3 region could be compensated for by replacement with HAdV-5 E4orf6; the resultant construct could replicate well in HEK293 cells. The enhanced Green Fluorescent Protein (eGFP) was inserted into SAdV E3 region and expressed at high level. One-step growth curve showed that the replication of the SAdVs with HAdV-5 E4orf6 substitution and E1/E3 deletion was similar to that of wild-type SAdVs in HEK293 cells, but the modified SAdVs were replication-deficient in A549 cells which lack HAdV-5 E1A and E1B. Finally, we demonstrated that GZ3-12 could infect cells expressing hCAR or hDSG2 receptors. The successful isolation, characterization, and modification of novel SAdVs provide a potentially important vaccine and gene therapy candidate and a new strategy for the rapid acquisition and development of non-HAdV-based alternative vectors for human health applications. IMPORTANCE Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells. [ABSTRACT FROM AUTHOR]

Genetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stacking of Multigenes (PSM) system was developed to assemble multiple transgenes into a single T-DNA. Combining the advantages of Gibson assembly and Gateway cloning, the PSM system uses an inverted pyramid stacking route and allows fast, flexible and efficient stacking of multiple genes into a binary vector. The PSM system contains two modular designed entry vectors (each containing two different attL sites and two selectable markers) and one Gateway-compatible destination vector (containing four attR sites and two negative selection markers). The target genes are primarily assembled into the entry vectors via two parallel rounds of Gibson assembly reactions. Then, the cargos in the entry constructs are integrated into the destination vector via a single tube Gateway LR reaction. To demonstrate PSM’s capabilities, four and nine gene expression cassettes were respectively assembled into the destination vector to generate two binary expression vectors. The transgenic analysis of these constructs in Arabidopsis demonstrated the reliability of the constructs generated by PSM. Due to its flexibility, simplicity and versatility, PSM has great potential for genetic engineering, synthetic biology and the improvement of multiple traits.

The construction of a full-length infectious clone, essential for molecular virological study and vaccine development, is quite a challenge for viruses with long genomes or possessing complex nucleotide sequence structures. Herein, we have constructed infectious clones of foot-and-mouth disease virus (FMDV) types O and A by joining each viral coding region with our pKLS3 vector in a single isothermal reaction using Gibson Assembly (GA). pKLS3 is a 4.3-kb FMDV minigenome. To achieve optimal conditions for the DNA joining, each FMDV coding sequence was divided into two overlapping fragments of approximately 3.8 and 3.2 kb, respectively. Both DNA fragments contain the introduced linker sequences for assembly with the linearized pKLS3 vector. FMDV infectious clones were produced upon directly transfecting the GA reaction into baby hamster kidney-21 (BHK-21) cells. After passing in BHK-21 cells, both rescued FMDVs (rO189 and rNP05) demonstrated growth kinetics and antigenicity similar to their parental viruses. Thus far, this is the first report on GA-derived, full-length infectious FMDV cDNA clones. This simple DNA assembly method and the FMDV minigenome would facilitate the construction of FMDV infectious clones and enable genetic manipulation for FMDV research and custom-made FMDV vaccine production. [ABSTRACT FROM AUTHOR]

Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developed for large plasmids by directly transforming two overlapping PCR fragments into Escherichia coli. This method successfully generated mutations for plasmids of 8.3 kb and 11.0 kb with high efficiencies. The method only requires Q5 DNA polymerase and DpnI, which greatly reduces costs. The procedure is simple, including PCR reaction, DpnI treatment and transformation. This simple, efficient and economical site-directed mutagenesis method for large plasmids is likely to be widely applied in the future.

Cotton blue disease (CBD) and atypical‐CBD are the most important viral diseases of cotton plants in the southern region of South America. Common and atypical strains of cotton leafroll dwarf virus (CLRDV and CLRDV‐at, respectively) are thought to be causative agents of CBD and atypical‐CBD, respectively. Inoculation of test plants via aphid vectors is difficult, as is determining strains via molecular diagnosis; accordingly, it is difficult for breeders to evaluate the effects of blue disease‐associated virus infections in cotton lineages. In the present study, we attempted to circumvent these difficulties by producing six full‐length cDNA infectious clones from CLRDV and CLRDV‐at strains using the Gibson Assembly protocol. For inoculation of the infectious clones, a vacuum chamber‐mediated agroinfiltration protocol was adapted and applied. Using this protocol, 90%–100% of cotton plants became infected with the clones, which was not possible via syringe‐based agroinfiltration. A genotyping protocol based on RT‐qPCR targeting a specific region of the virus P0 protein was also developed, allowing rapid differentiation of CLRDV and CLRDV‐at. Applying this protocol to 68 field samples revealed that CLRDV‐at was dominant (50%) over CLRDV (5.8%) in single virus infections. These preliminary results imply that CLRDV‐at might occupy the ecological niche of CLRDV in the cotton fields of Brazil. [ABSTRACT FROM AUTHOR]

In Brazil, the main viral disease of melon plant is severe yellowing disease called "Amarelão do Meloeiro," and a polerovirus, cucurbit aphid-borne yellows virus (CABYV) was considered one of the etiological agents. This virus is a recombinant strain originated from CABYV and unknown polerovirus. Due to unsuccessful mechanical inoculations of CABYV to host plants, the study of its biological characterization is hampered. Therefore, an infectious clone of the recombinant strain of CABYV was constructed using the Gibson Assembly technology. The full-length cDNA clones produced in this study showed to be infectious in three cucurbit species; melon (Cucumis melo), squash (a hybrid of Cucurbita maxima × C. moschata), and West Indian gherkin (Cucumis anguria) plants, but not in watermelon, cucumber, and zucchini plants. This insusceptibility of watermelon plants to the infectious clone corroborates the observation that this virus was never found in watermelon plants often located next to the infected melon plants. This infectious clone provides important tools for future study in developing resistant melon variety to CABYV infection. [ABSTRACT FROM AUTHOR]

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening., (© 2025 Wiley Periodicals LLC.)

Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.

The construction of a full-length infectious clone, essential for molecular virological study and vaccine development, is quite a challenge for viruses with long genomes or possessing complex nucleotide sequence structures. Herein, we have constructed infectious clones of foot-and-mouth disease virus (FMDV) types O and A by joining each viral coding region with our pKLS3 vector in a single isothermal reaction using Gibson Assembly (GA). pKLS3 is a 4.3-kb FMDV minigenome. To achieve optimal conditions for the DNA joining, each FMDV coding sequence was divided into two overlapping fragments of approximately 3.8 and 3.2 kb, respectively. Both DNA fragments contain the introduced linker sequences for assembly with the linearized pKLS3 vector. FMDV infectious clones were produced upon directly transfecting the GA reaction into baby hamster kidney-21 (BHK-21) cells. After passing in BHK-21 cells, both rescued FMDVs (rO189 and rNP05) demonstrated growth kinetics and antigenicity similar to their parental viruses. Thus far, this is the first report on GA-derived, full-length infectious FMDV cDNA clones. This simple DNA assembly method and the FMDV minigenome would facilitate the construction of FMDV infectious clones and enable genetic manipulation for FMDV research and custom-made FMDV vaccine production.

Summary: Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein–protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co‐transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all‐in‐one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single‐tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high‐throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS‐guided GA‐mediated AioFFP vector toolkit is cost‐effective, easy to use and will promote functional genomics research in plants. [ABSTRACT FROM AUTHOR]

Oncolytic adenoviruses have become ideal agents in the path toward treating cancer. Such viruses have been engineered to conditionally replicate in malignant cells in which certain signaling pathways have been disrupted. Other than such oncolytic properties, the viruses need to activate the immune system in order to sustain a long-term response. Therefore, oncolytic adenoviruses have been genetically modified to express various immune-stimulatory agents to achieve this. However, genetically modifying adenoviruses is very time consuming and labor intensive with the current available methods. In this paper, we describe a novel method we have called GAMER-Ad to genetically modify adenovirus genomes within 2 days. Our method entails the replacement of the gp19k gene in the E3 region with any given gene of interest (GOI) using Gibson Assembly avoiding the homologous recombination between the shuttle and the parental plasmid. In this manuscript as proof of concept we constructed and characterized three oncolytic adenoviruses expressing CXCL9, CXCL10, and interleukin-15 (IL-15). We demonstrate that our novel method is fast, reliable, and simple compared to other methods. We anticipate that our method will be used in the future to genetically engineer oncolytic but also other adenoviruses used for gene therapy as well.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses. [ABSTRACT FROM AUTHOR]

Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, limiting their efficiency and utility in high-throughput applications. More streamlined in silico tools allowing researchers to properly analyze and visualize influenza viral protein sequences with accurate nomenclature are necessary to improve antigen design and productivity. To address this need, we developed Librator, a system for analyzing and designing custom protein sequences of influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Within Librator's graphical interface, users can easily interrogate viral sequences and phylogenies, visualize antigen structures and conservation, mutate target residues and design custom antigens. Librator also provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible across related subtypes, thereby promoting reagent savings. Finally, the program facilitates single-cell immune profiling, epitope mapping of monoclonal antibodies and mosaic protein design. Using Librator - based antigen construction, we demonstrate that antigenicity can be readily transferred between HA molecules of H3, but not H1, lineage viruses. Altogether, Librator is a valuable tool for analyzing influenza virus HA and NA proteins and provides an efficient resource for optimizing recombinant influenza antigen synthesis. [ABSTRACT FROM AUTHOR]

Abstract An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.

New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

The discovery of the position effect variegation phenomenon and the subsequent comprehensive analysis of its molecular mechanisms led to understanding that the local chromatin composition has a dramatic effect on gene activity. To study this effect in a high-throughput mode and at the genome-wide level, the Thousands of Reporters Integrated in Parallel (TRIP) approach based on the usage of barcoded reporter gene constructs was recently developed. Here we describe the construction and quality checks of high-diversity barcoded plasmid libraries supposed to be used for high-throughput analysis of chromatin position effects in Drosophila cells. First, we highlight the critical parameters that should be considered in the generation of barcoded plasmid libraries and introduce a simple method to assess the diversity of random sequences (barcodes) of synthetic oligonucleotides using PCR amplification followed by Sanger sequencing. Second, we compare the conventional restriction-ligation method with the Gibson assembly approach for cloning barcodes into the same plasmid vector. Third, we provide optimized parameters for the construction of barcoded plasmid libraries, such as the vector : insert ratio in the Gibson assembly reaction and the voltage used for electroporation of bacterial cells with ligation products. We also compare different approaches to check the quality of barcoded plasmid libraries. Finally, we briefly describe alternative approaches that can be used for the generation of such libraries. Importantly, all improvements and modifications of the techniques described here can be applied to a wide range of experiments involving barcoded plasmid libraries.

Abstract Background The alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. The roles of vhs in HSV-1 and HSV-2 have been studied extensively in previous studies, however, relatively little is known about the vhs protein of PRV. Methods A novel method combining CRISPR/Cas9 and Gibson assembly was developed to generate UL41 null PRV variant. The properties of UL41 null PRV in vitro and in vivo were further characterized. And the vhs activity of UL41 protein of PRV variant was evaluated by luciferase assay, Western-blot and RT-qPCR. Results Gibson assembly based on homologous recombination can accomplish one-step insertion of viral DNA fragments into donor plasmids efficiently (> 80%). Cas9/gRNA further largely enhanced the efficiency of homologous recombination. Using this method we were able to rapidly generate the UL41 null and revertant viruses of PRV variant. Compared to wild type (JS-2012), the UL41 null virus showed significantly smaller plaques and lower titers in Vero cells and impaired lethality and neuroinvasion in mice. Further the UL41 protein from different PRV strains exhibited unequal vhs activity in vitro, which of JS-2012 showed significantly weaker vhs activity than that of European-American strains. In addition UL41 null virus can also significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be also involved in host shutoff. Conclusions CRISPR/Cas9 combined with Gibson assembly efficiently generated UL41 null PRV. Compared to wild type, UL41 null PRV showed impaired both replication capability in vitro and neuroinvasion in vivo. Further UL41 protein of PRV variant showed significantly weaker vhs activity than that of PRV SC (European-American-like strain), suggesting the deficiency of vhs activity by the PRV variant UL41 protein.

Abstract Background Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. Results We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb− 1. Conclusion We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time‐consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N‐ and C‐terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost‐intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes. [ABSTRACT FROM AUTHOR]

Aim: The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK‐15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. Methods and Results: We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin‐resistant overexpression strain was confirmed by PCR using the Apra‐F and Apra‐R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant. Conclusion: We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real‐time RT‐PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. Significance and Impact of the Study: Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes. [ABSTRACT FROM AUTHOR]

Abstract Background Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. Methods We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. Results T he current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision. Conclusion This method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods.

• Easy-to-use approaches are lacking for site-directed modification of adenovector (Ad). • Construct an intermediate plasmid for site-directed mutagenesis by DNA assembly. • Restore modified intermediate plasmid to adenoviral plasmid by restriction-ligation. • Combined DNA assembly and restriction-ligation can be used for Ad modification. Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus. [ABSTRACT FROM AUTHOR]

Summary: Transcription Activator‐Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time‐consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102‐nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single‐strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest. [ABSTRACT FROM AUTHOR]

• Preexisting neutralizing antibodies of common adenovirus like Ad5 limited its usefulness for the development of infectious disease vaccines. • To circumvent this obstacle, we generated an ad-hoc adenovirus vector Sad23L, to develop a strategy improving virus-propagating efficiency. • The procedure of constructing this novel vector took a single week, and recombinant adenovirus was packaged with high titer in HEK293 cells. • Sad23L with ZIKV prM-E genes induced a fairly robust immune response similar to Ad5-prM-E but no cross-reactive neutralizing antibody. Adenoviral vectors have been widely used for the development of infectious disease vaccines. However, the challenge of human adenoviral vector rooted from the predominant adenovirus serotype 5 strain limiting its usefulness by the widespread pre-existing neutralizing antibodies in recipients. To circumvent this obstacle, we generated an ad-hoc adenovirus vector in human or primates. Here, a chimeric simian adenoviral vector Sad23 was constructed consisting in deleting of E1 and E3 regions of the full-length simian adenovirus serotype 23 genome (SAdV23) by Gibson assembly. To improve Sad23 virus propagating efficiency, the E4 region open reading frame 6 (orf6) was replaced by the corresponding element of human adenovirus type 5 (Ad5), designated Sad23L. The procedure for cloning this novel vector took a single week, and recombinant adenovirus was packaged with high titer in HEK293 cells. To verify the ability of this novel adenoviral vector to deliver foreign genes, Zika virus (ZIKV) prM-E genes were used as target genes for antigen expression. Recombinant adenoviruses Sad23L-prM-E, Sad23-prM-E and Ad5-prM-E were intramuscularly inoculated into Ad5-eGFP none pre-exposed or pre-exposed mice, and the immune response to ZIKV prM-E was compared between vectors. Sad23L-prM-E induced a fairly robust immune response and maintained immunogenicity in Ad5 pre-exposed mice, which suggested that Ad5 pre-existing immunity did not affect Sad23L-prM-E immunization. These preliminary results suggest that the proposed rapid strategy was effective in constructing a new adenoviral vector platform (Sad23 L) usable for the development of human vaccines. [ABSTRACT FROM AUTHOR]

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes. In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors. [ABSTRACT FROM AUTHOR]

Summary: Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market. [ABSTRACT FROM AUTHOR]

A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.

The step catalyzed by terpene synthases is a well-recognized and significant bottleneck in engineered terpenoid bioproduction. Consequently, substantial efforts have been devoted towards increasing metabolic flux catalyzed by terpene synthases, employing strategies such as gene overexpression and protein engineering. Notably, numerous studies have demonstrated remarkable titer improvements by applying translational fusion, typically by fusing the terpene synthase with a prenyl diphosphate synthase that catalyzes the preceding step in the pathway. The main appeal of the translational fusion approach lies in its simplicity and orthogonality to other metabolic engineering tools. However, there is currently limited understanding of the underlying mechanism of flux enhancement, owing to the unpredictable and often protein-specific effects of translational fusion. In this chapter, we discuss practical considerations when engineering translationally fused terpene synthases, drawing insights from our experience and existing literature. We also provide detailed experimental workflows and protocols based on our previous work in budding yeast (Saccharomyces cerevisiae). Our intention is to encourage further research into the translational fusion of terpene synthases, anticipating that this will contribute mechanistic insights not only into the activity, behavior, and regulation of terpene synthases, but also of other enzymes., (Copyright © 2024. Published by Elsevier Inc.)

The production of geminiviral infectious clones provides a standardized inoculum for use in several host-virus studies. Geminiviruses present either one (monopartite) or two (bipartite) circular single-stranded DNA components, which commonly range from 2.6 to 2.8 kb. Cloning of a monomeric genome is useful for obtaining its precise sequence. For infectious clones, however, it is essential that more than one copy of the genome, more specifically of the origin of replication, is present in order to guarantee the production of full-length progeny DNA. Here, the complete process of preparing infectious geminiviral clones is described starting from the DNA extraction and selection of restriction endonucleases followed by two protocols for constructing dimeric clones: restriction endonuclease digestion and ligation (1) and Gibson Assembly (2)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract Viruses belonging to the genus Begomovirus (family Geminiviridae) have circular single-strand DNA genomes encapsidated into quasi-icosahedral particles, and are transmitted by whiteflies of the Bemisia tabaci complex. Biological and molecular properties of begomoviruses have been studied efficiently with infectious clones containing dimeric genomic components. However, current approaches employing enzymatic digestion and ligation to binary vectors are laborious, mostly due to many cloning steps or partial digestion by restriction enzyme. Here, an infectious clone of the bipartite begomovirus Bean golden mosaic virus (BGMV) was obtained using PCR and Gibson Assembly (GA). Common bean (Phaseolus vulgaris) seedlings displayed severe yellow mosaic and stunt symptoms 15 days after agroinoculation with DNA-A and DNA-B of BGMV. The approach based on PCR-GA protocol is a fast and useful tool to obtain infectious clones of a circular DNA plant virus. [ABSTRACT FROM AUTHOR]

Two members of the Benyviridae family and genus Benyvirus , Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV), possess identical genome organization, host range and high sequence similarity; they infect Beta vulgaris with variable symptom expression. In the US, mixed infections are described with limited information about viral interactions. Vectors suitable for agroinoculation of all genome components of both viruses were constructed by isothermal in vitro recombination. All 35S promoter-driven cDNA clones allowed production of recombinant viruses competent for Nicotiana benthamiana and Beta macrocarpa systemic infection and Polymyxa betae transmission and were compared to available BNYVV B-type clone. BNYVV and BSBMV RNA1 + 2 reassortants were viable and spread long-distance in N. benthamiana with symptoms dependent on the BNYVV type. Small genomic RNAs were exchangeable and systemically infected B. macrocarpa . These infectious clones represent a powerful tool for the identification of specific molecular host-pathogen determinants. [ABSTRACT FROM AUTHOR]

Humidimycin (MDN-0010) is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to class I lasso peptides, and is structurally related to siamycins, which have been shown to have strong antimicrobial activities against Gram-positive bacteria and to possess anti-HIV activity. Humidimycin was isolated from the strain Streptomyces humidus CA-100629, and was shown to synergize the activity of the fungal cell wall inhibitor caspofungin. In this work, the biosynthetic gene cluster of humidimycin was identified by genome mining of S. humidus CA-100629, cloned by Gibson assembly, and heterologously expressed.

Background: Recombinant DNA technology is today a fundamental tool for virtually all biological research fields. Among the many techniques available for the construction of a "custom DNA" molecule, the isothermal in vitro assembly, or Gibson assembly, allows for an efficient, one-step, scarless recombination-based assembly. Results: Here, we apply and characterize the use of Gibson assembly for the deletion of DNA sequences around a DNA cut. This method, that we named "Gibson Deletion", can be used to easily substitute or delete one or more restriction sites within a DNA molecule. We show that Gibson Deletion is a viable method to delete up to 100 nucleotides from the DNA ends of a cleavage site. In addition, we found that Gibson Deletion can be performed using single strand DNA with the same efficiency as using double strand DNA molecules. Conclusions: Gibson Deletion is a novel, easy and convenient application of isothermal in vitro assembly, that performs with high efficiency and can be implemented for a broad range of applications. [ABSTRACT FROM AUTHOR]

Allelic exchange mutagenesis that relies on RecA -mediated homologous recombination up- and downstream from the targeted gene is a generalizable method of site-specific bacterial gene knock-out and knock-in. However, generation of a mutagenic DNA construct (alternative allele flanked by regions surrounding the gene target) and subsequent mutant selection are laborious procedures. Here we demonstrate allelic exchange knock-out facilitated by Gibson Assembly in Streptococcus iniae . Gibson Assembly allows rapid construction of a large allelic exchange cassette simultaneous with cloning, as well as rapid reconstruction of complete recombinant vector sequence when required. Additionally, we show that during two-step mutant selection, absence of recombination at one of the homologous regions (single cross-over) might be rapidly detected by colony PCR of meroploid clones and resolved by extension/shifting of corresponding sequence in DNA construct. The combination of Gibson Assembly for mutagenic DNA construction/redesign with colony PCR screening of meroploids to detect recombination at both sides of the exchange target may significantly accelerate generation of chromosomal mutants in a wide range of bacterial taxa. [ABSTRACT FROM AUTHOR]

The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8 bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57-66 bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. Methods: We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. Results: The current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision. Conclusion: This method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods. [ABSTRACT FROM AUTHOR]

Genetic modification of large DNA fragments (gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity. In this study, we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction (PCR) targeting combined with Gibson assembly. In this strategy, the biosynthetic genes for peptidyl moieties (HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid (CPOAA) from the polyoxin biosynthetic gene cluster to generate a ~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette. The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic, polynik A, was obtained and verified. This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production. [ABSTRACT FROM AUTHOR]

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean. [ABSTRACT FROM AUTHOR]

Traditional methods for the assembly of functionalised DNA structures, involving enzyme restriction and modification, present difficulties when working with small DNA fragments (<100 bp), in part due to a lack of control over enzymatic action during the DNA modification process. This limits the design flexibility and range of accessible DNA structures. Here, we show that these limitations can be overcome by introducing chemical modifications into the DNA that spatially restrict enzymatic activity. This approach, sterically controlled nuclease enhanced (SCoNE) DNA assembly, thereby circumvents the size limitations of conventional Gibson assembly (GA) and allows the preparation of well-defined, functionalised DNA structures with multiple probes for specific analytes, such as IL-6, procalcitonin (PCT), and a biotin reporter group. Notably, when using the same starting materials, conventional GA under typical conditions fails. We demonstrate successful analyte capture based on standard and modified sandwich ELISA and also show how the inclusion of biotin probes provides additional functionality for product isolation., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene., Competing Interests: The author(s) declare that there are no conflicts of interest., (© 2023 The Authors.)