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1.
Nature ; 585(7823): 129-134, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848250

RESUMO

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.


Assuntos
Simulação por Computador , Genes Sintéticos/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Biologia Sintética , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Condutividade Elétrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canais Iônicos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Engenharia de Proteínas , Estrutura Secundária de Proteína , Solubilidade , Água/química
2.
Int J Phytoremediation ; 22(12): 1269-1277, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32449363

RESUMO

Bioremediation with genetically modified microalgae is becoming an alternative to remove metalloids and metals such as cadmium, a contaminant produced in industrial processes and found in domestic waste. Its removal is important in several countries including Mexico, where the San Luis Potosi region has elevated levels of it. We generated a construct with a synthetic gene for γ-glutamylcysteine synthetase and employed it in the chloroplast transformation of Chlamydomonas reinhardtii. In dose-response kinetics with media containing from 1 to 20 mg/L of cadmium, both the transplastomic clone and the wild-type strain grew similarly, but the former removed up to 32% more cadmium. While the growth of both decreased with higher concentrations of cadmium, the transplastomic clone removed 20 ± 9% more than the wild-type strain. Compared to the wild-type strain, in the transplastomic clone the activity of glutathione S-transferase and the intracellular glutathione increased up to 2.1 and 1.9 times, respectively, in media with 2.5 and 10 mg/mL of cadmium. While 20 mg/L of cadmium inhibited the growth of both, the transplastomic clone gradually duplicated. These results confirm the expression of the synthetic gene gshA in the transformed strain as revealed in its increased removal uptake and metabolic response.


Assuntos
Chlamydomonas reinhardtii/genética , Biodegradação Ambiental , Cádmio , Genes Sintéticos , Glutamato-Cisteína Ligase/genética , México
3.
Nat Chem Biol ; 16(6): 695-701, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32251409

RESUMO

Growth-mediated feedback between synthetic gene circuits and host organisms leads to diverse emerged behaviors, including growth bistability and enhanced ultrasensitivity. However, the range of possible impacts of growth feedback on gene circuits remains underexplored. Here we mathematically and experimentally demonstrated that growth feedback affects the functions of memory circuits in a network topology-dependent way. Specifically, the memory of the self-activation switch is quickly lost due to the growth-mediated dilution of the circuit products. Decoupling of growth feedback reveals its memory, manifested by its hysteresis property across a broad range of inducer concentration. On the contrary, the toggle switch is more refractory to growth-mediated dilution and can retrieve its memory after the fast-growth phase. The underlying principle lies in the different dependence of active and repressive regulations in these circuits on the growth-mediated dilution. Our results unveil the topology-dependent mechanism on how growth-mediated feedback influences the behaviors of gene circuits.


Assuntos
Proteínas de Escherichia coli/genética , Redes Reguladoras de Genes/genética , Genes Sintéticos/genética , Simulação por Computador , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Modelos Genéticos , Imagem Óptica
4.
Nat Commun ; 11(1): 868, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054834

RESUMO

Synthetic biology, genome engineering and directed evolution offer innumerable tools to expedite engineering of strains for optimising biosynthetic pathways. One of the most radical is SCRaMbLE, a system of inducible in vivo deletion and rearrangement of synthetic yeast chromosomes, diversifying the genotype of millions of Saccharomyces cerevisiae cells in hours. SCRaMbLE can yield strains with improved biosynthetic phenotypes but is limited by screening capabilities. To address this bottleneck, we combine automated sample preparation, an ultra-fast 84-second LC-MS method, and barcoded nanopore sequencing to rapidly isolate and characterise the best performing strains. Here, we use SCRaMbLE to optimise yeast strains engineered to produce the triterpenoid betulinic acid. Our semi-automated workflow screens 1,000 colonies, identifying and sequencing 12 strains with between 2- to 7-fold improvement in betulinic acid titre. The broad applicability of this workflow to rapidly isolate improved strains from a variant library makes this a valuable tool for biotechnology.


Assuntos
Genes Sintéticos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triterpenos/metabolismo , Biotecnologia , Cromatografia Líquida , Cromossomos Fúngicos , Evolução Molecular Direcionada , Biblioteca Gênica , Estudos de Associação Genética , Engenharia Genética , Testes Genéticos , Genoma Fúngico , Espectrometria de Massas , Recombinação Genética , Biologia Sintética
5.
Nat Commun ; 11(1): 867, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054832

RESUMO

Alzheimer's disease (AD) is defined by progressive neurodegeneration, with oligomerization and aggregation of amyloid-ß peptides (Aß) playing a pivotal role in its pathogenesis. In recent years, the yeast Saccharomyces cerevisiae has been successfully used to clarify the roles of different human proteins involved in neurodegeneration. Here, we report a genome-wide synthetic genetic interaction array to identify toxicity modifiers of Aß42, using yeast as the model organism. We find that FMN1, the gene encoding riboflavin kinase, and its metabolic product flavin mononucleotide (FMN) reduce Aß42 toxicity. Classic experimental analyses combined with RNAseq show the effects of FMN supplementation to include reducing misfolded protein load, altering cellular metabolism, increasing NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios and increasing resistance to oxidative stress. Additionally, FMN supplementation modifies Htt103QP toxicity and α-synuclein toxicity in the humanized yeast. Our findings offer insights for reducing cytotoxicity of Aß42, and potentially other misfolded proteins, via FMN-dependent cellular pathways.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Mononucleotídeo de Flavina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Genes Sintéticos , Genoma Fúngico , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Modelos Genéticos , Mutação , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Dobramento de Proteína , Proteólise , RNA-Seq , Riboflavina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
6.
PLoS One ; 15(2): e0229542, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32107503

RESUMO

BACKGROUND: The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. METHODOLOGY/PRINCIPAL FINDINGS: We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. CONCLUSIONS/SIGNIFICANCE: It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.


Assuntos
Antígenos de Helmintos , Schistosoma japonicum/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/genética , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/genética , Esôfago/imunologia , Genes de Helmintos , Genes Sintéticos , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Macaca mulatta , Camundongos , Análise Serial de Proteínas , Coelhos , Ratos , Schistosoma japonicum/genética , Esquistossomose Japônica/imunologia , Esquistossomose Japônica/prevenção & controle , Vacinas Sintéticas/genética
7.
J Neurosci ; 40(7): 1405-1426, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31915257

RESUMO

BDNF signaling via its transmembrane receptor TrkB has an important role in neuronal survival, differentiation, and synaptic plasticity. Remarkably, BDNF is capable of modulating its own expression levels in neurons, forming a transcriptional positive feedback loop. In the current study, we have investigated this phenomenon in primary cultures of rat cortical neurons using overexpression of dominant-negative forms of several transcription factors, including CREB, ATF2, C/EBP, USF, and NFAT. We show that CREB family transcription factors, together with the coactivator CBP/p300, but not the CRTC family, are the main regulators of rat BDNF gene expression after TrkB signaling. CREB family transcription factors are required for the early induction of all the major BDNF transcripts, whereas CREB itself directly binds only to BDNF promoter IV, is phosphorylated in response to BDNF-TrkB signaling, and activates transcription from BDNF promoter IV by recruiting CBP. Our complementary reporter assays with BDNF promoter constructs indicate that the regulation of BDNF by CREB family after BDNF-TrkB signaling is generally conserved between rat and human. However, we demonstrate that a nonconserved functional cAMP-responsive element in BDNF promoter IXa in humans renders the human promoter responsive to BDNF-TrkB-CREB signaling, whereas the rat ortholog is unresponsive. Finally, we show that extensive BDNF transcriptional autoregulation, encompassing all major BDNF transcripts, occurs also in vivo in the adult rat hippocampus during BDNF-induced LTP. Collectively, these results improve the understanding of the intricate mechanism of BDNF transcriptional autoregulation.SIGNIFICANCE STATEMENT Deeper understanding of stimulus-specific regulation of BDNF gene expression is essential to precisely adjust BDNF levels that are dysregulated in various neurological disorders. Here, we have elucidated the molecular mechanisms behind TrkB signaling-dependent BDNF mRNA induction and show that CREB family transcription factors are the main regulators of BDNF gene expression after TrkB signaling. Our results suggest that BDNF-TrkB signaling may induce BDNF gene expression in a distinct manner compared with neuronal activity. Moreover, our data suggest the existence of a stimulus-specific distal enhancer modulating BDNF gene expression.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Córtex Cerebral/citologia , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Transcrição Genética/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Córtex Cerebral/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Retroalimentação Fisiológica , Feminino , Genes Dominantes , Genes Reporter , Genes Sintéticos , Hipocampo/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor trkB/fisiologia , Proteínas Recombinantes/farmacologia , Elementos de Resposta , Especificidade da Espécie , Transdução Genética
8.
Nat Methods ; 17(1): 93-100, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31768062

RESUMO

Single-cell genetic screens can be incredibly powerful, but current high-throughput platforms do not track dynamic processes, and even for non-dynamic properties they struggle to separate mutants of interest from phenotypic outliers of the wild-type population. Here we introduce SIFT, single-cell isolation following time-lapse imaging, to address these limitations. After imaging and tracking individual bacteria for tens of consecutive generations under tightly controlled growth conditions, cells of interest are isolated and propagated for downstream analysis, free of contamination and without genetic or physiological perturbations. This platform can characterize tens of thousands of cell lineages per day, making it possible to accurately screen complex phenotypes without the need for barcoding or genetic modifications. We applied SIFT to identify a set of ultraprecise synthetic gene oscillators, with circuit variants spanning a 30-fold range of average periods. This revealed novel design principles in synthetic biology and demonstrated the power of SIFT to reliably screen diverse dynamic phenotypes.


Assuntos
Separação Celular/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos , Rastreamento de Células/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Biblioteca Gênica , Genes Sintéticos , Processamento de Imagem Assistida por Computador , Microfluídica/métodos
9.
Biochem Soc Trans ; 47(6): 1795-1804, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31803907

RESUMO

Cell-to-cell variability originating, for example, from the intrinsic stochasticity of gene expression, presents challenges for designing synthetic gene circuits that perform robustly. Conversely, synthetic biology approaches are instrumental in uncovering mechanisms underlying variability in natural systems. With a focus on reducing noise in individual genes, the field has established a broad synthetic toolset. This includes noise control by engineering of transcription and translation mechanisms either individually, or in combination to achieve independent regulation of mean expression and its variability. Synthetic feedback circuits use these components to establish more robust operation in closed-loop, either by drawing on, but also by extending traditional engineering concepts. In this perspective, we argue that major conceptual advances will require new theory of control adapted to biology, extensions from single genes to networks, more systematic considerations of origins of variability other than intrinsic noise, and an exploration of how noise shaping, instead of noise reduction, could establish new synthetic functions or help understanding natural functions.


Assuntos
Células , Redes Reguladoras de Genes , Genes Sintéticos , Regulação da Expressão Gênica , Biossíntese de Proteínas , Processos Estocásticos , Biologia Sintética , Transcrição Genética
11.
Med Sci (Paris) ; 35(10): 753-760, 2019 Oct.
Artigo em Francês | MEDLINE | ID: mdl-31625897

RESUMO

The past decade has seen vast improvements in DNA synthesis and assembly methods. The creation of synthetic DNA molecules is becoming easier and more affordable, such that entire chromosomes can now be synthesized. These advances mark the beginning of synthetic genomics, a new discipline interested in the construction of complete genomes tailored for the study and application of biological systems. From viral genome synthesis to the reconstruction of the yeast 16 chromosomes, we discuss the main discoveries, the regulations and ethical considerations along with the potential of this emerging discipline for the future.


Assuntos
Cromossomos , DNA/síntese química , Genes Sintéticos , Previsões , Biologia Sintética/ética , Biologia Sintética/métodos , Biologia Sintética/tendências
12.
Med Sci (Paris) ; 35(10): 761-770, 2019 Oct.
Artigo em Francês | MEDLINE | ID: mdl-31625898

RESUMO

The development of synthetic genomics (SG) allowed the emergence of several groundbreaking techniques including the synthesis, assembly and engineering of whole bacterial genomes. The successful implantation of those methods, which culminated in the creation of JCVI-syn3.0 the first nearly minimal bacterium with a synthetic genome, mainly results from the use of the yeast Saccharomyces cerevisiae as a transient host for bacterial genome replication and modification. Another method played a key role in the resounding success of this project: bacterial genome transplantation (GT). GT consists in the transfer of bacterial genomes cloned in yeast, back into a cellular environment suitable for the expression of their genetic content. While successful using many mycoplasma species, a complete understanding of the factors governing GT will most certainly help unleash the power of the entire SG pipeline to other genetically intractable bacteria.


Assuntos
Genes Sintéticos , Genoma Bacteriano/genética , Genômica/métodos , Saccharomyces cerevisiae , Biologia Sintética/métodos
13.
Nat Commun ; 10(1): 4801, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641136

RESUMO

Improving efficacy of oncolytic virotherapy remains challenging due to difficulty increasing specificity and immune responses against cancer and limited understanding of its population dynamics. Here, we construct programmable and modular synthetic gene circuits to control adenoviral replication and release of immune effectors selectively in hepatocellular carcinoma cells in response to multiple promoter and microRNA inputs. By performing mouse model experiments and computational simulations, we find that replicable adenovirus has a superior tumor-killing efficacy than non-replicable adenovirus. We observe a synergistic effect on promoting local lymphocyte cytotoxicity and systematic vaccination in immunocompetent mouse models by combining tumor lysis and secretion of immunomodulators. Furthermore, our computational simulations show that oncolytic virus which encodes immunomodulators can exert a more robust therapeutic efficacy than combinatorial treatment with oncolytic virus and immune effector. Our results provide an effective strategy to engineer oncolytic adenovirus, which may lead to innovative immunotherapies for a variety of cancers.


Assuntos
Adenoviridae/genética , Genes Sintéticos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Animais , Carcinoma Hepatocelular/terapia , Feminino , Células HEK293 , Células Hep G2 , Humanos , Imunoterapia/métodos , Neoplasias Hepáticas/terapia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Curr Protoc Mol Biol ; 127(1): e89, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31237422

RESUMO

Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.


Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Marcação de Genes/métodos , Genes Sintéticos/genética , Oryza/genética , Ativação Transcricional , Sistemas CRISPR-Cas/genética , Genes de Plantas/genética , Regiões Promotoras Genéticas , Protoplastos/metabolismo , RNA Guia/genética
17.
Am J Respir Cell Mol Biol ; 61(6): 747-754, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31184507

RESUMO

Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/uso terapêutico , Fibrose Cística/terapia , Dependovirus/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Animais , Animais Recém-Nascidos , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/administração & dosagem , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Elementos de DNA Transponíveis , Técnicas de Inativação de Genes , Genes Sintéticos , Humanos , Regiões Promotoras Genéticas , Staphylococcus aureus , Suínos , Traqueia/metabolismo , Traqueia/microbiologia , Integração Viral
18.
Curr Opin Biotechnol ; 59: 130-140, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31128430

RESUMO

Synthetic biology efforts began in simple single-cell systems, which were relatively easy to manipulate genetically (Cameron et al., 2014). The field grew exponentially in the last two decades, and one of the latest frontiers are synthetic developmental programs for multicellular mammalian systems (Black et al., 2017; Wieland and Fussenegger, 2012) to genetically control features such as patterning or morphogenesis. These programs rely on engineered cell-cell communications, multicellular gene regulatory networks and effector genes. Here, we contextualize the first of these synthetic developmental programs, examine molecular and computational tools that can be used to generate next generation versions, and present the general logic that underpins these approaches. These advances are exciting as they represent a novel way to address both control and understanding in the field of developmental biology and tissue development (Elowitz and Lim, 2010; Velazquez et al., 2018; White et al., 2018; Morsut, 2017). This field is just at the beginning, and it promises to be of major interest in the upcoming years of biomedical research.


Assuntos
Genes Sintéticos , Biologia Sintética , Animais , Comunicação Celular , Redes Reguladoras de Genes , Mamíferos
19.
Methods Enzymol ; 621: 17-30, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128777

RESUMO

The budding yeast Saccharomyces cerevisiae has been widely utilized in fermentative production since ancient times. Several approaches for modification of yeast traits have been developed, including mutagenesis, protoplast fusion, and genetic modification. Crossbreeding provides an attractive means to improve and combine strain traits based on sexual reproduction. Common crossbreeding strategies require the isolation of MATa and MATα haploids via sporulation, as most of parental yeasts are MATa/α diploids and unable to mate directly. Unfortunately, many yeast strains used in industry exhibit low sporulation rates resulting in limited crossbreeding efficiency and numerous technical challenges. Here, we review the construction of synthetic gene expression circuits as a means to provide alternative methods for sporulation for yeast crossbreeding. These methods enable researchers to convert the sequence of the MAT locus and subsequently acquire crossbreds via mating of isolated yeast strains. The purpose of this chapter is to provide a basic guide for researchers who are attempting to expand the variety of yeast resources using the sexual reproduction machinery of yeast.


Assuntos
Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Genes Sintéticos , Saccharomyces cerevisiae/genética , Genes Fúngicos , Haploidia , Técnicas Microbiológicas , Saccharomyces cerevisiae/fisiologia
20.
Nucleic Acids Res ; 47(13): 6714-6725, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31127286

RESUMO

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.


Assuntos
Proteínas de Arabidopsis/fisiologia , RNA Polimerase II/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequenciamento de Cromatina por Imunoprecipitação , DNA de Plantas/genética , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Sintéticos , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Mensageiro/biossíntese , RNA de Plantas/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Elongação da Transcrição Genética , Sítio de Iniciação de Transcrição
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