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2.
J Microbiol Biotechnol ; 29(5): 776-784, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31030455

RESUMO

Polyhydroxybutyrate (PHB), the most well-known polyhydroxyalkanoate, is a bio-based, biodegradable polymer that has the potential to replace petroleum-based plastics. Lignocellulose hydrolysate, a non-edible resource, is a promising substrate for the sustainable, fermentative production of PHB. However, its application is limited by the generation of inhibitors during the pretreatment processes. In this study, we investigated the feasibility of PHB production in E. coli in the presence of inhibitors found in lignocellulose hydrolysates. Our results show that the introduction of PHB synthetic genes (bktB, phaB, and phaC from Ralstonia eutropha H16) improved cell growth in the presence of the inhibitors such as furfural, 4-hydroxybenzaldehyde, and vanillin, suggesting that PHB synthetic genes confer resistance to these inhibitors. In addition, increased PHB production was observed in the presence of furfural as opposed to the absence of furfural, suggesting that this compound could be used to stimulate PHB production. Our findings indicate that PHB production using lignocellulose hydrolysates in recombinant E. coli could be an innovative strategy for cost-effective PHB production, and PHB could be a good target product from lignocellulose hydrolysates, especially glucose.


Assuntos
Aclimatação/genética , Escherichia coli/genética , Furaldeído/efeitos adversos , Genes Sintéticos/genética , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Proteínas de Bactérias/genética , Cupriavidus necator/genética , Resistência a Medicamentos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Hordeum/enzimologia , Lignina/metabolismo , Pinus/enzimologia , Poaceae/embriologia
3.
Mol Biotechnol ; 60(8): 608-620, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29855997

RESUMO

Designing the expression cassettes with desired properties remains the most important consideration of gene engineering technology. One of the challenges for predictive gene expression is the modeling of synthetic gene switches to regulate one or more target genes which would directly respond to specific chemical, environmental, and physiological stimuli. Assessment of natural promoter, high-throughput sequencing, and modern biotech inventory aided in deciphering the structure of cis elements and molding the native cis elements into desired synthetic promoter. Synthetic promoters which are molded by rearrangement of cis motifs can greatly benefit plant biotechnology applications. This review gives a glimpse of the manual in vivo gene regulation through synthetic promoters. It summarizes the integrative design strategy of synthetic promoters and enumerates five approaches for constructing synthetic promoters. Insights into the pattern of cis regulatory elements in the pursuit of desirable "gene switches" to date has also been reevaluated. Joint strategies of bioinformatics modeling and randomized biochemical synthesis are addressed in an effort to construct synthetic promoters for intricate gene regulation.


Assuntos
Expressão Gênica/genética , Genes Sintéticos/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas/genética , Engenharia Genética/métodos , Plantas/genética
4.
Science ; 361(6400)2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29930090

RESUMO

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.


Assuntos
Proteínas de Ligação a DNA/química , Domínios e Motivos de Interação entre Proteínas , Imagem Individual de Molécula/métodos , Fatores de Transcrição/química , Transcrição Genética , Ativação Transcricional , Linhagem Celular Tumoral , Genes Sintéticos , Humanos , Regiões Operadoras Genéticas , Ligação Proteica , RNA Polimerase II/química
5.
PLoS Comput Biol ; 14(4): e1006055, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29614119

RESUMO

Recent studies have demonstrated how the competition for the finite pool of available gene expression factors has important effect on fundamental gene expression aspects. In this study, based on a whole-cell model simulation of translation in S. cerevisiae, we evaluate for the first time the expected effect of mRNA levels fluctuations on translation due to the finite pool of ribosomes. We show that fluctuations of a single gene or a group of genes mRNA levels induce periodic behavior in all S. cerevisiae translation factors and aspects: the ribosomal densities and the translation rates of all S. cerevisiae mRNAs oscillate. We numerically measure the oscillation amplitudes demonstrating that fluctuations of endogenous and heterologous genes can cause a significant fluctuation of up to 50% in the steady-state translation rates of the rest of the genes. Furthermore, we demonstrate by synonymous mutations that oscillating the levels of mRNAs that experience high ribosomal occupancy (e.g. ribosomal "traffic jam") induces the largest impact on the translation of the S. cerevisiae genome. The results reported here should provide novel insights and principles related to the design of synthetic gene expression circuits and related to the evolutionary constraints shaping gene expression of endogenous genes.


Assuntos
Modelos Genéticos , Biossíntese de Proteínas , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Códon/genética , Biologia Computacional , Simulação por Computador , Evolução Molecular , Expressão Gênica , Genes Sintéticos , Genoma Fúngico , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Cinética , Método de Monte Carlo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Ribossomos/metabolismo
6.
Adv Exp Med Biol ; 1029: 49-68, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29542080

RESUMO

Ascidians possess relatively small and compact genomes. This feature enables us to easily isolate cis-regulatory DNAs of genes of interest. Particularly, cis-regulatory DNAs of genes showing tissue- or cell-type-specific expression are routinely used for the artificial induction of gene expression. This strategy helps us to label cells, tissues, and organs of interest, and to investigate gene functions through overexpression, ectopic expression, and the disruption of functions by dominant-negative forms. Thus, cis-regulatory DNAs provide a powerful tool for tissue-specific genetic manipulation in studies of ascidian development and physiology. This chapter summarizes the types of cis-regulatory DNAs as a genetic manipulation tool, describes the methods used for isolating cis-regulatory DNAs, and provide reported examples of the use of cis-regulatory DNAs as molecular tools for investigating gene functions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Urocordados/genética , Animais , Linhagem da Célula , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Embrião não Mamífero/citologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Sintéticos , Técnicas Genéticas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Larva , Especificidade de Órgãos , Regiões Promotoras Genéticas , Transcrição Genética , Transgenes , Urocordados/embriologia , Urocordados/crescimento & desenvolvimento
7.
Adv Exp Med Biol ; 1029: 153-164, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29542088

RESUMO

Ascidians are increasingly being used as a system for investigating cell biology during development. The extreme genetic and cellular simplicity of ascidian embryos in combination with superior experimental tractability make this an ideal system for in vivo analysis of dynamic cellular processes. Transgenic approaches to cellular and sub-cellular analysis of ascidian development have begun to yield new insights into the mechanisms regulating developmental signaling and morphogenesis. This chapter focuses on the targeted expression of fusion proteins in ascidian embryos and how this technique is being deployed to garner new insights into the cell biology of development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Urocordados/genética , Animais , Animais Geneticamente Modificados , Ciclo Celular , Movimento Celular/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Genes Reporter , Genes Sintéticos , Larva , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Tubo Neural/embriologia , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/genética , Fuso Acromático , Transgenes , Urocordados/citologia , Urocordados/embriologia , Urocordados/crescimento & desenvolvimento
8.
Nat Commun ; 9(1): 776, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29472537

RESUMO

Modern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. Here we report a synthetic genetic system implementing population-based AND-logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations.


Assuntos
Bactérias/genética , Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Sintéticos , Engenharia Genética , Regiões Promotoras Genéticas , Biologia Sintética/instrumentação , Biologia Sintética/métodos , Proteínas Virais/metabolismo
9.
Science ; 359(6376)2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29439214

RESUMO

Gene and engineered-cell therapies promise to treat diseases by genetically modifying cells to carry out therapeutic tasks. Although the field has had some success in treating monogenic disorders and hematological malignancies, current approaches are limited to overexpression of one or a few transgenes, constraining the diseases that can be treated with this approach and leading to potential concerns over safety and efficacy. Synthetic gene networks can regulate the dosage, timing, and localization of gene expression and therapeutic activity in response to small molecules and disease biomarkers. Such "programmable" gene and engineered-cell therapies will provide new interventions for incurable or difficult-to-treat diseases.


Assuntos
Engenharia Celular/métodos , Terapia Baseada em Transplante de Células e Tecidos , Técnicas de Reprogramação Celular , Engenharia Genética/métodos , Terapia Genética , Biologia Sintética/métodos , DNA/genética , Expressão Gênica , Redes Reguladoras de Genes , Genes Sintéticos , Humanos , RNA/genética , Proteínas Recombinantes de Fusão , Transgenes
10.
Proc Natl Acad Sci U S A ; 115(5): 992-997, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29343642

RESUMO

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.


Assuntos
Imunoterapia/métodos , Neoplasias/terapia , Animais , Fenômenos Biomecânicos , Sinalização do Cálcio , Genes Sintéticos , Engenharia Genética , Técnicas Genéticas , Células HEK293 , Humanos , Canais Iônicos/genética , Canais Iônicos/imunologia , Células Jurkat , Mecanotransdução Celular/genética , Mecanotransdução Celular/imunologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Biologia Sintética , Linfócitos T/imunologia , Ultrassom
11.
Science ; 359(6373): 343-347, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29301959

RESUMO

Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene's assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in Escherichia coli We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.


Assuntos
Genes Sintéticos , Proteínas/fisiologia , Emulsões , Escherichia coli/genética , Técnicas de Inativação de Genes , Genes Essenciais , Teste de Complementação Genética , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Proteínas/genética , Biologia Sintética/métodos
12.
Biochemistry ; 57(5): 764-771, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29283561

RESUMO

CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kd and kcat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy-ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis.


Assuntos
17-alfa-Hidroxipregnenolona/metabolismo , 17-alfa-Hidroxiprogesterona/metabolismo , Androstenodiona/biossíntese , Desidroepiandrosterona/biossíntese , Esteroide 17-alfa-Hidroxilase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Catálise , Domínio Catalítico , Sequência Conservada , Genes Sintéticos , Humanos , Ligações de Hidrogênio , Mamíferos/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Esteroide 17-alfa-Hidroxilase/química , Esteroide 17-alfa-Hidroxilase/genética , Especificidade por Substrato
13.
Dev Biol ; 433(2): 262-275, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29198566

RESUMO

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.


Assuntos
Ambystoma mexicanum/fisiologia , Membro Anterior/fisiologia , Regulação da Expressão Gênica , Vetores Genéticos/genética , Nucleopolyhedrovirus/genética , Regeneração/fisiologia , Transdução Genética , Ambystoma mexicanum/genética , Amputação , Animais , Perfilação da Expressão Gênica , Genes Reporter , Genes Sintéticos , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiologia , Proteínas de Homeodomínio/fisiologia , Humanos , Glicoproteínas de Membrana/fisiologia , Mesoderma/citologia , Proteínas Recombinantes/metabolismo , Regeneração/genética , Transgenes , Proteínas do Envelope Viral/fisiologia , Cicatrização/genética , Cicatrização/fisiologia
14.
Curr Genet ; 64(2): 327-333, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28983660

RESUMO

Full genome recoding, or rewriting codon meaning, through chemical synthesis of entire bacterial chromosomes has become feasible in the past several years. Recoding an organism can impart new properties including non-natural amino acid incorporation, virus resistance, and biocontainment. The estimated cost of construction that includes DNA synthesis, assembly by recombination, and troubleshooting, is now comparable to costs of early stage development of drugs or other high-tech products. Here, we discuss several recently published assembly methods and provide some thoughts on the future, including how synthetic efforts might benefit from the analysis of natural recoding processes and organisms that use alternative genetic codes.


Assuntos
DNA/biossíntese , Evolução Molecular , Genes Sintéticos/genética , Código Genético/genética , Códon/genética , DNA/genética , Escherichia coli/genética , Engenharia Genética , Genoma Bacteriano/genética
15.
Nat Biomed Eng ; 2(6): 399-415, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011195

RESUMO

In living organisms, naturally evolved sensors that constantly monitor and process environmental cues trigger corrective actions that enable the organisms to cope with changing conditions. Such natural processes have inspired biologists to construct synthetic living sensors and signalling pathways, by repurposing naturally occurring proteins and by designing molecular building blocks de novo, for customized diagnostics and therapeutics. In particular, designer cells that employ user-defined synthetic gene circuits to survey disease biomarkers and to autonomously re-adjust unbalanced pathological states can coordinate the production of therapeutics, with controlled timing and dosage. Furthermore, tailored genetic networks operating in bacterial or human cells have led to cancer remission in experimental animal models, owing to the network's unprecedented specificity. Other applications of designer cells in infectious, metabolic and autoimmune diseases are also being explored. In this Review, we describe the biomedical applications of synthetic gene circuits in major disease areas, and discuss how the first genetically engineered devices developed on the basis of synthetic-biology principles made the leap from the laboratory to the clinic.


Assuntos
Doenças Transmissíveis , Redes Reguladoras de Genes/genética , Genes Sintéticos/genética , Biologia Sintética , Nanomedicina Teranóstica , Animais , Controle de Doenças Transmissíveis , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/terapia , Engenharia Genética , Humanos , Camundongos
16.
BMC Genomics ; 18(1): 990, 2017 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-29281970

RESUMO

BACKGROUND: Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown. RESULTS: We engineered the tTAV system in Drosophila melanogaster and investigated the effects of tTAV genome integration locus on the whole fly transcriptome during larval and adult life stages in four transgenic fly strains using gene expression microarrays. We found that while there were widespread effects on the transcriptome, the gene expression differences after removal of tetracycline were not consistent between integration sites. No specific region of the genome was affected, no common set of genes or pathways, nor did the integration site affect the transcripts in cis. CONCLUSION: Although the positive feedback tTAV system is effective at killing insect larvae regardless of where it is inserted in the genome, it does not exhibit a specific, consistent transcriptional signature. Instead, each insertion site is associated with broad, but different, transcriptional effects. Our results suggest that lethality may not be caused by a direct effect on transcription of a set of key genes or pathways. Instead, we propose that rather than a specific action of a tTAV protein, it is the stochastic transcriptional effects specific to each insertion site that contribute to the tTAV-induced mortality.


Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genes Sintéticos , Transcrição Genética , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Retroalimentação Fisiológica , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da Espécie , Transcriptoma
17.
J Immunol ; 199(12): 3959-3971, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29101311

RESUMO

Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction.


Assuntos
Apresentação do Antígeno , Células Apresentadoras de Antígenos/imunologia , Autoantígenos/imunologia , Deleção Clonal/imunologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Fatores de Transcrição/imunologia , Animais , Autoantígenos/biossíntese , Autoantígenos/genética , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Genes Sintéticos , Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/biossíntese , Ovalbumina/genética , Ovalbumina/imunologia , Regiões Promotoras Genéticas , Ratos , Organismos Livres de Patógenos Específicos , Timo/citologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transgenes
18.
BMC Syst Biol ; 11(1): 112, 2017 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-29169395

RESUMO

BACKGROUND: The pursuit of standardization and reliability in synthetic biology has achieved, in recent years, a number of advances in the design of more predictable genetic parts for biological circuits. However, even with the development of high-throughput screening methods and whole-cell models, it is still not possible to predict reliably how a synthetic genetic construct interacts with all cellular endogenous systems. This study presents a genome-wide analysis of how the expression of synthetic genes is affected by systematic perturbations of cellular functions. We found that most perturbations modulate expression indirectly through an effect on cell size, putting forward the existence of a generic Size-Expression interaction in the model prokaryote Escherichia coli. RESULTS: The Size-Expression interaction was quantified by inserting a dual fluorescent reporter gene construct into each of the 3822 single-gene deletion strains comprised in the KEIO collection. Cellular size was measured for single cells via flow cytometry. Regression analyses were used to discriminate between expression-specific and gene-specific effects. Functions of the deleted genes broadly mapped onto three systems with distinct primary influence on the Size-Expression map. Perturbations in the Division and Biosynthesis (DB) system led to a large-cell and high-expression phenotype. In contrast, disruptions of the Membrane and Motility (MM) system caused small-cell and low-expression phenotypes. The Energy, Protein synthesis and Ribosome (EPR) system was predominantly associated with smaller cells and positive feedback on ribosome function. CONCLUSIONS: Feedback between cell growth and gene expression is widespread across cell systems. Even though most gene disruptions proximally affect one component of the Size-Expression interaction, the effect therefore ultimately propagates to both. More specifically, we describe the dual impact of growth on cell size and gene expression through cell division and ribosomal content. Finally, we elucidate aspects of the tight control between swarming, gene expression and cell growth. This work provides foundations for a systematic understanding of feedbacks between genetic and physiological systems.


Assuntos
Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano , Proteínas de Bactérias/biossíntese , Deleção de Genes , Redes Reguladoras de Genes , Genes Reporter , Genes Sintéticos , Ensaios de Triagem em Larga Escala
19.
Nat Commun ; 8(1): 1191, 2017 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084946

RESUMO

The nuclease-deactivated variant of CRISPR-Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable orthologous dCas9 or Cpf1 effector proteins, thus opening options for multidimensional control of functional activities through combinations of orthogonal, drug-tunable artificial transcription factors.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endonucleases/genética , Genes Sintéticos/genética , RNA Guia/genética , Animais , Proteínas de Bactérias/metabolismo , Células CHO , Proteína 9 Associada à CRISPR , Cricetinae , Cricetulus , Endonucleases/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
PLoS One ; 12(10): e0186633, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29040308

RESUMO

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.


Assuntos
Proteínas de Bactérias/metabolismo , Códon/química , Enzimas de Restrição do DNA/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/química , Thermus thermophilus/enzimologia , Proteínas de Bactérias/genética , Composição de Bases , Sequência de Bases , Clonagem Molecular , Códon/metabolismo , Enzimas de Restrição do DNA/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Genes Sintéticos , Temperatura Alta , Cinética , Metiltransferases/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Thermus thermophilus/genética
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