Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Synth Biol (Oxf) ; 7(1): ysac018, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36285185

RESUMO

We describe an experimental campaign that replicated the performance assessment of logic gates engineered into cells of Saccharomyces cerevisiae by Gander et al. Our experimental campaign used a novel high-throughput experimentation framework developed under Defense Advanced Research Projects Agency's Synergistic Discovery and Design program: a remote robotic lab at Strateos executed a parameterized experimental protocol. Using this protocol and robotic execution, we generated two orders of magnitude more flow cytometry data than the original experiments. We discuss our results, which largely, but not completely, agree with the original report and make some remarks about lessons learned. Graphical Abstract.

2.
PLOS Glob Public Health ; 2(2): e0000185, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36962187

RESUMO

Usability is an overlooked aspect of implementing lab-based assays, particularly novel assays in low-resource-settings. Esoteric instructions can lead to irreproducible test results and patient harm. To address these issues, we developed a software application based on "Aquarium", a laboratory-operating system run on a computer tablet that provides step-by-step digital interactive instructions, protocol management, and sample tracking. Aquarium was paired with a near point-of-care HIV drug resistance test, "OLA-Simple", that detects mutations associated with virologic failure. In this observational study we evaluated the performance of Aquarium in guiding untrained users through the multi-step laboratory protocol with little supervision. To evaluate the training by Aquarium software we conducted a feasibility study in a laboratory at Coptic Hope Center in Nairobi, Kenya. Twelve volunteers who were unfamiliar with the kit performed the test on blinded samples (2 blood specimens; 5 codons/sample). Steps guided by Aquarium included: CD4+ T-Cell separation, PCR, ligation, detection, and interpretation of test results. Participants filled out a short survey regarding their demographics and experience with the software and kit. None of the laboratory technicians had prior experience performing CD4+ separation and 7/12 had no experience performing laboratory-based molecular assays. 12/12 isolated CD4+ T cells from whole blood with yields comparable to isolations performed by trained personnel. The OLA-Simple workflow was completed by all, with genotyping results interpreted correctly by unaided-eye in 108/120 (90%) and by software in 116/120 (97%) of codons analyzed. In the surveys, participants favorably assessed the use of software guidance. The Aquarium digital instructions enabled first-time users in Kenya to complete the OLA-simple kit workflow with minimal training. Aquarium could increase the accessibility of laboratory assays in low-resource-settings and potentially standardize implementation of clinical laboratory tests.

3.
Nat Commun ; 8: 15459, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28541304

RESUMO

Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses. We built a combinatorial library of NOR gates that directly convert guide RNA (gRNA) inputs into gRNA outputs, enabling the gates to be 'wired' together. We constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Modelling predicted the NOR gates have effectively zero transcriptional leak explaining the limited signal degradation in the circuits. Our approach enabled the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, cellular decision-making systems.


Assuntos
Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Montagem e Desmontagem da Cromatina/genética , Genes Fúngicos , Genes Sintéticos , Engenharia Genética , Lógica , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Fúngico/genética , RNA Guia de Cinetoplastídeos/genética , Biologia Sintética/métodos
4.
ACS Synth Biol ; 3(11): 832-8, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25350266

RESUMO

Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast.


Assuntos
Criptocromos/metabolismo , Optogenética/métodos , Fitocromo/metabolismo , Transcrição Gênica/genética , Criptocromos/química , Criptocromos/genética , Dimerização , MAP Quinases Reguladas por Sinal Extracelular , Fitocromo/química , Fitocromo/genética , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Leveduras/genética
5.
Nat Commun ; 5: 4925, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25233328

RESUMO

The Arabidopsis photoreceptor cryptochrome 2 (CRY2) was previously used as an optogenetic module, allowing spatiotemporal control of cellular processes with light. Here we report the development of a new CRY2-derived optogenetic module, 'CRY2olig', which induces rapid, robust, and reversible protein oligomerization in response to light. Using this module, we developed a novel protein interaction assay, Light-Induced Co-clustering, that can be used to interrogate protein interaction dynamics in live cells. In addition to use probing protein interactions, CRY2olig can also be used to induce and reversibly control diverse cellular processes with spatial and temporal resolution. Here we demonstrate disrupting clathrin-mediated endocytosis and promoting Arp2/3-mediated actin polymerization with light. These new CRY2-based approaches expand the growing arsenal of optogenetic strategies to probe cellular function.


Assuntos
Análise por Conglomerados , Optogenética/métodos , Mapeamento de Interação de Proteínas/métodos , Proteína 2 Relacionada a Actina/metabolismo , Actinas/química , Animais , Arabidopsis/genética , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clatrina/química , Citoesqueleto/metabolismo , Citosol/metabolismo , Endocitose , Células HEK293 , Humanos , Luz , Mutação , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
6.
Dev Biol ; 395(1): 84-95, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25176044

RESUMO

Peroxisome biogenesis disorders (PBD) are autosomal recessive disorders in humans characterized by skeletal, eye and brain abnormalities. Despite the fact that neurological deficits, including peripheral nervous system (PNS) defects, can be observed at birth in some PBD patients including those with PEX10 mutations, the embryological basis of the PNS defects is unclear. Using a forward genetic screen, we identified a mouse model for Pex10 deficiency that exhibits neurological abnormalities during fetal development. Homozygous Pex10 mutant mouse embryos display biochemical abnormalities related to a PBD deficiency. During late embryogenesis, Pex10 homozygous mutant mice experience progressive loss of movement and at birth they become cyanotic and die shortly thereafter. Homozygous Pex10 mutant fetuses display decreased integrity of axons and synapses, over-extension of axons in the diaphragm and decreased Schwann cell numbers. Our neuropathological, molecular and electrophysiological studies provide new insights into the embryological basis of the PNS deficits in a PBD model. Our findings identify PEX10 function, and likely other PEX proteins, as an essential component of the spinal locomotor circuit.


Assuntos
Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Transtornos Peroxissômicos/genética , Receptores Citoplasmáticos e Nucleares/genética , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Humanos , Imuno-Histoquímica , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Atividade Motora/genética , Placa Motora/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Doenças do Sistema Nervoso Periférico/embriologia , Doenças do Sistema Nervoso Periférico/metabolismo , Peroxinas , Transtornos Peroxissômicos/embriologia , Transtornos Peroxissômicos/metabolismo , Peroxissomos/metabolismo , Peroxissomos/ultraestrutura , Receptores Citoplasmáticos e Nucleares/metabolismo , Nervo Isquiático/embriologia , Nervo Isquiático/metabolismo , Homologia de Sequência de Aminoácidos , Doenças da Medula Espinal/embriologia , Doenças da Medula Espinal/genética , Doenças da Medula Espinal/metabolismo
7.
Curr Protoc Cell Biol ; 64: 17.16.1-20, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25181301

RESUMO

Genetically encoded actuators that allow control of protein-protein interactions using light, termed 'optical dimerizers', are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and the coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use in controlling transcription, protein localization, and protein secretion using light. Additionally, we provide instructions and software for constructing a pulse-controlled LED device for use in experiments requiring extended light treatments.


Assuntos
Proteínas de Arabidopsis/química , Proteínas Cromossômicas não Histona/química , Criptocromos/química , Luz , Multimerização Proteica , Animais , Proteínas de Arabidopsis/genética , Células COS , Chlorocebus aethiops , Proteínas Cromossômicas não Histona/genética , Criptocromos/genética , Células HeLa , Humanos
8.
Biol Cell ; 105(2): 59-72, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23157573

RESUMO

Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.


Assuntos
Optogenética/métodos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos da radiação , Animais , Humanos , Luz
9.
Genetics ; 192(3): 831-42, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22923379

RESUMO

Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.


Assuntos
Alelos , Estabilidade Enzimática/genética , Genes Reporter , Ensaios de Triagem em Larga Escala , Dobramento de Proteína , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática/efeitos dos fármacos , Estudos de Associação Genética , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína/efeitos dos fármacos , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade , Transaminases/química , Transaminases/genética , Transaminases/metabolismo
10.
J Biol Chem ; 287(26): 22165-72, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22577138

RESUMO

Plant photoreceptors transduce environmental light cues to downstream signaling pathways, regulating a wide array of processes during growth and development. Two major plant photoreceptors with critical roles in photomorphogenesis are phytochrome B (phyB), a red/far-red absorbing photoreceptor, and cryptochrome 1 (CRY1), a UV-A/blue photoreceptor. Despite substantial genetic evidence for cross-talk between phyB and CRY1 pathways, a direct interaction between these proteins has not been observed. Here, we report that Arabidopsis phyB interacts directly with CRY1 in a light-dependent interaction. Surprisingly, the interaction is light-dissociated; CRY1 interacts specifically with the dark/far-red (Pr) state of phyB, but not with the red light-activated (Pfr) or the chromophore unconjugated form of the enzyme. The interaction is also regulated by light activation of CRY1; phyB Pr interacts only with the unstimulated form of CRY1 but not with the photostimulated protein. Further studies reveal that a small domain extending from the photolyase homology region (PHR) of CRY1 regulates the specificity of the interaction with different conformational states of phyB. We hypothesize that in plants, the phyB/CRY1 interaction may mediate cross-talk between the red/far-red- and blue/UV-sensing pathways, enabling fine-tuning of light responses to different spectral inputs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Criptocromos/metabolismo , Criptocromos/fisiologia , Fitocromo B/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Modelos Biológicos , Células Fotorreceptoras/metabolismo , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Raios Ultravioleta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...