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Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.
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Regulação da Expressão Gênica , Super Intensificadores , Transcrição Gênica , alfa-Globinas , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , alfa-Globinas/genéticaRESUMO
Yeast research is entering into a new period of scholarship, with new scientific tools, new questions to ask and new issues to consider. The politics of emerging and critical technology can no longer be separated from the pursuit of basic science in fields, such as synthetic biology and engineering biology. Given the intensifying race for technological leadership, yeast research is likely to attract significant investment from government, and that it offers huge opportunities to the curious minded from a basic research standpoint. This article provides an overview of new directions in yeast research with a focus on Saccharomyces cerevisiae, and places these trends in their geopolitical context. At the highest level, yeast research is situated within the ongoing convergence of the life sciences with the information sciences. This convergent effect is most strongly pronounced in areas of AI-enabled tools for the life sciences, and the creation of synthetic genomes, minimal genomes, pan-genomes, neochromosomes and metagenomes using computer-assisted design tools and methodologies. Synthetic yeast futures encompass basic and applied science questions that will be of intense interest to government and nongovernment funding sources. It is essential for the yeast research community to map and understand the context of their research to ensure their collaborations turn global challenges into research opportunities.
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Centromeres are the genomic regions that organize and regulate chromosome behaviours during cell cycle, and their variations are associated with genome instability, karyotype evolution and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.
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Centrômero , Melhoramento Vegetal , Humanos , Centrômero/genética , Cromossomos de Plantas/genética , Plantas/genética , Epigenômica , BiotecnologiaRESUMO
A range of game-changing biodigital and biodesign technologies are coming of age all around us, transforming our world in complex ways that are hard to predict. Not a day goes by without news of how data-centric engineering, algorithm-driven modelling, and biocyber technologies-including the convergence of artificial intelligence, machine learning, automated robotics, quantum computing, and genome editing-will change our world. If we are to be better at expecting the unexpected in the world of wine, we need to gain deeper insights into the potential and limitations of these technological developments and advances along with their promise and perils. This article anticipates how these fast-expanding bioinformational and biodesign toolkits might lead to the creation of synthetic organisms and model systems, and ultimately new understandings of biological complexities could be achieved. A total of four future frontiers in wine yeast research are discussed in this article: the construction of fully synthetic yeast genomes, including minimal genomes; supernumerary pan-genome neochromosomes; synthetic metagenomes; and synthetic yeast communities. These four concepts are at varying stages of development with plenty of technological pitfalls to overcome before such model chromosomes, genomes, strains, and yeast communities could illuminate some of the ill-understood aspects of yeast resilience, fermentation performance, flavour biosynthesis, and ecological interactions in vineyard and winery settings. From a winemaker's perspective, some of these ideas might be considered as far-fetched and, as such, tempting to ignore. However, synthetic biologists know that by exploring these futuristic concepts in the laboratory could well forge new research frontiers to deepen our understanding of the complexities of consistently producing fine wines with different fermentation processes from distinctive viticultural terroirs. As the saying goes in the disruptive technology industry, it take years to create an overnight success. The purpose of this article is neither to glorify any of these concepts as a panacea to all ills nor to crucify them as a danger to winemaking traditions. Rather, this article suggests that these proposed research endeavours deserve due consideration because they are likely to cast new light on the genetic blind spots of wine yeasts, and how they interact as communities in vineyards and wineries. Future-focussed research is, of course, designed to be subject to revision as new data and technologies become available. Successful dislodging of old paradigms with transformative innovations will require open-mindedness and pragmatism, not dogmatism-and this can make for a catch-22 situation in an archetypal traditional industry, such as the wine industry, with its rich territorial and socio-cultural connotations.
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Vinho , Inteligência Artificial , Metodologias Computacionais , Fermentação , Teoria Quântica , Saccharomyces cerevisiae/genética , Vinho/análiseRESUMO
Nucleic acids are the basic units of deoxyribonucleic acid (DNA) sequencing. Every organism demonstrates different DNA sequences with specific nucleotides. It reveals the genetic information carried by a particular DNA segment. Nucleic acid sequencing expresses the evolutionary changes among organisms and revolutionizes disease diagnosis in animals. This paper proposes a generative adversarial networks (GAN) model to create synthetic nucleic acid sequences of the cat genome tuned to exhibit specific desired properties. We obtained the raw sequence data from Illumina next generation sequencing. Various data preprocessing steps were performed using Cutadapt and DADA2 tools. The processed data were fed to the GAN model that was designed following the architecture of Wasserstein GAN with gradient penalty (WGAN-GP). We introduced a predictor and an evaluator in our proposed GAN model to tune the synthetic sequences to acquire certain realistic properties. The predictor was built for extracting samples with a promoter sequence, and the evaluator was built for filtering samples that scored high for motif-matching. The filtered samples were then passed to the discriminator. We evaluated our model based on multiple metrics and demonstrated outputs for latent interpolation, latent complementation, and motif-matching. Evaluation results showed our proposed GAN model achieved 93.7% correlation with the original data and produced significant outcomes as compared to existing models for sequence generation.
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Adenosina Desaminase , Processamento de Imagem Assistida por Computador , DNA , Processamento de Imagem Assistida por Computador/métodos , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERSâ¢ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoVâ¢ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.
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Doenças Transmissíveis Emergentes/virologia , Coronavirus/genética , DNA Complementar/genética , Síndrome Respiratória Aguda Grave/virologia , Northern Blotting , Western Blotting , Células Cultivadas , Primers do DNA/genética , Dipeptidil Peptidase 4/metabolismo , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Proteínas Luminescentes , Oriente Médio , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia , Ligação Viral , Replicação Viral/fisiologia , Proteína Vermelha FluorescenteRESUMO
Introduction: Organisms engineered with synthetic genes and genomes have the potential to play critical roles to address important issues in the environment, human health, and manufacturing. Engineered genetic biocontainment technologies are needed to manage the risks of unintended consequences when deploying these biological systems in consultation with the biosafety and biosecurity communities. Metrics measuring genetic biocontainment and a methodology to apply them are required to determine which genetic biocontainment technologies warrant further development for real-world applications. In this study, we develop and apply a systems analysis of the current technical landscape using expert opinion and a metric-based scoring system resulting in a semiquantitative comparative assessment of genetic biocontainment technologies in microorganisms. Methods: Genetic biocontainment technologies were evaluated according to multiple metrics, falling into two broad classes: feasibility and applicability. Specific genetic biocontainment example scenarios and generalized categories were scored with these metrics. Gap analysis was carried out, indicating particular areas where genetic biocontainment can be improved. Results: Metric analysis scoring of feasibility and applicability enabled prioritization of genetic biocontainment technologies for real-world applications. Gap analysis showed that technology readiness and containment stability scored low for a number of scenarios and categories, indicating a general need for further development before they can be ready for deployment. Conclusion: Developing an assessment framework with defined metrics produced a straightforward system for evaluating genetic biocontainment strategies intended for various real-world applications. Use of the methodology also provided insights into existing gaps in genetic biocontainment strategies, and by altering the metrics, can be applied to other biotechnologies.
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Synthetic genomes were designed based on an understanding of natural genomic information, offering an opportunity to engineer and investigate biological systems on a genome-wide scale. Currently, the designer version of the M. mycoides genome and the E. coli genome, as well as most of the S. cerevisiae genome, have been synthesized, and through the cycles of design-build-test and the following engineering of synthetic genomes, many fundamental questions of genome biology have been investigated. In this review, we summarize the use of synthetic genome engineering to explore the structure and function of genomes, and highlight the unique values of synthetic genomics.
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Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.
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The engineering of viral genomes facilitates both fundamental and applied research on viruses. However, the multiloci manipulation of DNAs of viruses with large DNA genomes, such as baculoviruses, herpesviruses, and poxviruses, is technically challenging, particularly for highly homologous or repetitive sequences. Homologous regions (hrs) have multiple copies in many large DNA viruses and play pivotal roles in the viral life cycle. Here, we used synthetic biology to investigate the fundamental function of baculoviral hrs by conducting multiloci manipulation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA that contains eight hrs scattered in the genome. Using transformation-associated recombination in yeast, we generated recombinant AcMNPV genomes in which we deleted all hrs or retained a single hr (hr1, hr2, or hr3). Infectious viruses were rescued after transfecting the synthetic viral genomes into host cells, and their replication features were characterized. The results demonstrated that deletion of all hrs severely compromised viral DNA replication and progeny production, whereas retaining only a single hr was essential for efficient viral DNA replication and progeny production. The synthetic virus with hr2 or hr3 showed a growth curve similar to that of the parental virus. Transcriptomic analysis revealed that hr1, hr2, and hr3 could enhance gene transcription within a surrounding region of 14.6 kb, 13.8 kb, and 29.8 kb, respectively. Overall, this study revealed the advantages of synthetic biology in multiloci engineering and functional studies of large DNA viruses. In addition, our findings on hrs will be helpful for the design and improvement of baculovirus-based expression vectors.
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Baculoviridae , Replicação Viral , Animais , Baculoviridae/genética , Replicação do DNA/genética , DNA Viral/genética , Spodoptera/genética , Replicação Viral/genéticaRESUMO
Baculoviruses have been widely used as expression vectors. However, numerous genes in the baculoviral genome are non-essential for cellular infection and protein expression, making the optimisation of baculovirus expression vectors possible. We used a synthetic biological method to reduce the number of genes in a partial region of the autograph californica multiple nucleopolyhedrovirus (AcMNPV), the most widely used baculovirus expression vector. The C1 region of the AcMNPV is 46.4 kb and is subdivided into B1, B2, and B3 fragments. We first designed modified B1, B2, and B3 fragments by deleting the non-essential genes, and then synthesised complete viral genomes containing either individual modified B fragments or joint modified B fragments through transformation-related recombination in yeast. The synthetic genomes were then transfected into Sf9 cells to rescue the progeny viruses and test their infectivity. The design-build-test cycle was repeated until the ultimately rescued virus could produce progeny viruses efficiently. Finally, AcMNPV-Syn-mC1-1.1 by deleting approximately 17.2 kb, including 20 ORFs, in the C1 region, was obtained. This is essential to the synthesis of a minimal AcMNPV genome that can generate infectious progeny viruses and can be further used to optimise the foundation of baculovirus expression vectors.
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Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/genética , Spodoptera , Baculoviridae , Células Sf9 , Replicação Viral/genéticaRESUMO
Parvovirus B19 (B19V) is a human pathogenic virus of clinical relevance, characterized by a selective tropism for erythroid progenitor cells in bone marrow. Relevant information on viral characteristics and lifecycle can be obtained from experiments involving engineered genetic systems in appropriate in vitro cellular models. Previously, a B19V genome of defined consensus sequence was designed, synthesized and cloned in a complete and functional form, able to replicate and produce infectious viral particles in a producer/amplifier cell system. Based on such a system, we have now designed and produced a derived B19V minigenome, reduced to a replicon unit. The genome terminal regions were maintained in a form able to sustain viral replication, while the internal region was clipped to include only the left-side genetic set, containing the coding sequence for the functional NS1 protein. Following transfection in UT7/EpoS1 cells, this minigenome still proved competent for replication, transcription and production of NS1 protein. Further, the B19V minigenome was able to complement B19-derived, NS1-defective genomes, restoring their ability to express viral capsid proteins. The B19V genome was thus engineered to yield a two-component system, with complementing functions, providing a valuable tool for studying viral expression and genetics, suitable to further engineering for purposes of translational research.
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Genoma Viral , Parvovirus B19 Humano/genética , Parvovirus B19 Humano/fisiologia , Replicon , Linhagem Celular , Clonagem Molecular , Engenharia Genética , Humanos , Transcrição Gênica , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
Naturally occurring structural variations (SVs) are a considerable source of genomic variation that can reshape the 3D architecture of chromosomes. Controllable methods aimed at introducing the complex SVs and their related molecular mechanisms have remained farfetched. In this study, an SV-prone yeast strain was developed using Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) technology with two synthetic chromosomes, namely synV and synX. The biosynthesis of astaxanthin is used as a readout and a proof of concept for the application of SVs in industries. Our findings showed that complex SVs, including a pericentric inversion and a trans-chromosome translocation between synV and synX, resulted in two neo-chromosomes and a 2.7-fold yield of astaxanthin. Also, genetic targets were mapped, which resulted in a higher astaxanthin yield, thus demonstrating the SVs' ability to reorganize genetic information along the chromosomes. The rational design of trans-chromosome translocation and pericentric inversion enabled precise induction of these phenomena. Collectively, this study provides an effective tool to not only accelerate the directed genome evolution but also to reveal the mechanistic insight of complex SVs for altering phenotypes.
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Saccharomyces cerevisiae , Translocação Genética , Cromossomos , Evolução Molecular , Genoma , Humanos , Saccharomyces cerevisiae/genéticaRESUMO
Genome synthesis endows scientists the ability of de novo creating genomes absent in nature, by thorough redesigning DNA sequences and introducing numerous custom features. However, the genome synthesis is a labor- and time-consuming work, and thus it is a challenge to verify and quantify the synthetic genome rapidly and precisely. Thus, specific DNA sequences different from native genomic sequences are designed into synthetic genomes during synthesis, namely genomic markers. Genomic markers can be easily detected by PCR reaction, whole-genome sequencing (WGS) and a variety of methods to identify the synthetic genome from native one. Here, we review types and applications of genomic markers utilized in synthetic genomes, with the hope of providing a guidance for future works.
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In synthetic biology, biological cells and processes are dismantled and reassembled to make novel systems that do useful things. Designs are encoded by deoxyribonucleic acid (DNA); DNA makes biological (bio-)parts; bioparts are combined to make devices; devices are built into biological systems. Computers are used at all stages of the Design-Build-Test-Learn cycle, from mathematical modelling through to the use of robots for the automation of assembly and experimentation. Synthetic biology applies engineering principles of standardisation, modularity, and abstraction, enabling fast prototyping and the ready exchange of designs between synthetic biologists working around the world. Like toy building blocks, compatible modular designs enable bioparts to be combined and optimised easily; biopart specifications are shared in open registries. Synthetic biology is made possible due to major advances in DNA sequencing and synthesis technologies, and through knowledge gleaned in the field of systems biology. Systems biology aims to understand biology across scales, from the molecular and cellular, up to tissues and organisms, and describes cells as complex information-processing systems. By contrast, synthetic biology seeks to design and build its own systems. Applications of synthetic biology are wide-ranging but include impacting healthcare to improve diagnosis and make better treatments for disease; it seeks to improve the environment by finding novel ways to clean up pollution, make industrial processes for chemical synthesis sustainable, and remove the need for damaging farming practices by making better fertilisers. Synthetic biology has the potential to change the way we live and help us to protect the future of our planet.
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Biologia Sintética , Biologia de Sistemas , AgriculturaRESUMO
Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome. Recently, we generated a synthetic baculovirus AcMNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we report the modification of AcMNPV-WIV-Syn1 into a novel bacmid, AcBac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a LacZ:attTn7 and egfp cassette was constructed, and recombined with a linearized AcMNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid AcBac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the attTn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that AcBac-Syn can be readily used for foreign gene insertion and expression. AcBac-Syn has several advantages over the conventional AcMNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration; its DNA copy numbers could be induced to a higher level in E. coli; and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.
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Baculoviridae , Escherichia coli , Animais , Baculoviridae/genética , Vírus de DNA , Escherichia coli/genética , Genes Reporter , Spodoptera , Replicação ViralRESUMO
The Synthetic Genome Summer Course was convened with the aim of teaching a wide range of researchers the theory and practical skills behind recent advances in synthetic biology and synthetic genome science, with a focus on Sc2.0, the synthetic yeast genome project. Through software workshops, tutorials and research talks from leading members of the field, the 30 attendees learnt about relevant principles and techniques that they were then able to implement first-hand in laboratory-based practical sessions. Participants SCRaMbLEd semi-synthetic yeast strains to diversify heterologous pathways, used automation to build combinatorial pathway libraries and used CRISPR to debug fitness defects caused by synthetic chromosome design changes. Societal implications of synthetic chromosomes were explored and industrial stakeholders discussed synthetic biology from a commercial standpoint. Over the 5 days, participants gained valuable insight and acquired skills to aid them in future synthetic genome research.
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Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.
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Genes Sintéticos , Engenharia Genética , Genoma , Biologia Sintética , Genoma/genética , Genômica , Modelos Biológicos , Análise de SequênciaRESUMO
Synthetic viruses provide a powerful platform to delve deeper into the nature and function of viruses as well as to engineer viruses with novel properties. So far, most synthetic viruses have been RNA viruses (<30 kb) and small DNA viruses, such as bacteriophage phiX174. Baculoviruses contain a large circular dsDNA genome of 80-180 kb and have been used as biocontrol agents and protein expression vectors. Here, we report on the first synthesis of a baculovirus based on the type species Autographa californica nucleopolyhedrovirus, AcMNPV, by a combination of PCR and transformation-associated recombination in yeast. The synthetic genome, designated AcMNPV-WIV-Syn1, is 145â¯299 bp comprising the complete genome of AcMNPV except for the hr4a locus that was replaced with an â¼11.5 kb cassette of bacterial and yeast artificial chromosomal elements and an egfp gene. Sf9 insect cells were transfected with AcMNPV-WIV-Syn1 DNA and progeny virus was examined by electron microscopy, and assayed in one-step growth curves and oral infectivity. The results conclusively showed that the rescued virus AcMNPV-WIV-Syn1 had structural and biological properties comparable to the parental virus. We validated a proof of concept that a bona fide baculovirus can be synthesized. The new platform allows manipulation at any or multiple loci and will facilitate future studies such as identifying the minimal baculovirus genome and construction of better expression vectors. This is the largest DNA virus synthesized so far, and its success is likely to be the impetus to stimulate the fields of other large DNA viruses such as herpesviruses and poxviruses.
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Nucleopoliedrovírus/genética , Biologia Sintética/métodos , Animais , Cromossomos Artificiais/genética , Vírus de DNA/genética , DNA Viral/genética , Reação em Cadeia da Polimerase , Células Sf9RESUMO
Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus.