This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.

1. Preparação da biblioteca metagenômica com PNP-GESS <ol><li> Construir uma biblioteca metagenômica em E. coli com um vector fosmid usando um kit de produção fosmid biblioteca de acordo com o protocolo do fabricante 5. </li><li> Alíquota de 100 ul da biblioteca, para armazenamento a -70 ° C, que é uma fonte de células de biblioteca metagenomic. Nota: A densidade óptica de uma amostra medida a um comprimento de onda de 600 nm (OD 600) deste estoque biblioteca é de aproximadamente 100. </li><li> Descongelar 100 ul da biblioteca metagenômico estoque em gelo e em inocular um balão de 500 ml contendo 50 ml de meio Luria-Bertani (LB) e 12,5 ug / ml de cloranfenicol seguido de 37 ° C de incubação, durante 2 h. </li><li> Colher as células num tubo de 50 ml por centrifugação a 1000 xg durante 20 min a 4 ° C. </li><li> Ressuspender o sedimento rapidamente em 50 ml de água destilada gelada (DW) e centrifugar a 1000 xg durante 20 min a 4 ° C novamente. </li><li> Resuspend o sedimento em 50 ul DW gelada com 10% (v / v) de glicerol. Utilizar 50 ul desta alíquota de células para electroporação. </li><li> Coloque a mistura de células electrocompetentes de 50 ul e pGESS (E135K) 5 de DNA (100 ng) numa cuvete de electroporação arrefecido em gelo e electroporar (1,8 kV / cm, 25 mF) a mistura. </li><li> adicionar rapidamente 1 ml de super caldo ideal com catabólica repressão médio (SOC) e voltar a suspender as células suavemente. </li><li> Transferir as células para um tubo de 14 ml de fundo redondo, usando uma pipeta e incubar a 37 ° C durante 1 h. </li><li> Espalhe 500 ul das células recuperadas numa LB 20 x 20 cm placa quadrada contendo 12,5 ug / ml de cloranfenicol e 50 jig / ml de ampicilina. Incubar a placa a 30 ° C durante 12 h. </li><li> Raspe as colónias usando um raspador de células e recolher células em um tubo cônico de 50 ml utilizando meios de armazenamento de células gelada. </li><li> Centrifugar a 1.000 xg durante 20 min a 4 ° C. Ressuspender o sedimento em 10 ml de mídia de armazenamento de células geladaspara chegar a um OD 600 de 100. </li><li> Aliquota de 20 uL de células para armazenamento a -70 ° C. </li></ol> 2. Remoção falsos positivos a partir da biblioteca metagenômica <ol><li> Descongelar as células ações metagenomic biblioteca (do passo 1.13) contendo fosmid DNA metagenômica e pGESS (E135K) em gelo. </li><li> Inocular 10 ml de células em 2 ml de LB contendo 50 ug / ml de ampicilina e 12,5 jig / ml de cloranfenicol em um tubo de fundo redondo de 14 ml. Incubar a 37 ° C com agitação a 200 rpm durante 4 h. </li><li> Enquanto isso, ligue a máquina de FACS e abra o software FACS padrão. Utilize as seguintes definições: diâmetro da ponta do bico, 70 mm; Adiante Área de Dispersão (-A FSC) sensibilidade, 300 amplificação V-logarítmica; Side Area Scatter (-A SSC) Sensibilidade, 350 amplificação V-logarítmica; Área fluoresceína isotiocianato (FITC-A) sensibilidade, a amplificação de 450 V-logarítmica; parâmetro de patamar, FSC-Um valor 5. Nota: As configurações mais comuns são fornecidaspara o dispositivo de FACS mencionado na tabela de materiais. Ajustar as configurações para outros dispositivos de FACS. </li><li> Dilui-se as células a partir de bibliotecas metagenomic Passo 2.2 pela adição de 5 ul da amostra para um tubo de fundo redondo de 5 ml contendo 1 ml de PBS. </li><li> Colocar a amostra biblioteca diluída para o porto de carga do instrumento FACS e clique no botão de carga no Aquisição Painel do software FACS. </li><li> Ajustar a taxa de evento para 1.000 - 1.500 eventos / seg clicando e controlar os botões Taxa de fluxo no painel. </li><li> Criar log escalado FSC-A vs. log escalado SSC-Um gráfico de dispersão em uma planilha global, clicando no botão gráfico de pontos na barra de ferramentas. Ajuste o portão de dispersão R1 para abranger os eventos singlet (população bacteriana) usando o botão Polígono Gate na barra de ferramentas. </li><li> Traçar um histograma com a contagem de células vs. log escalado FITC-A na planilha clicando no botão histograma. Em seguida, ajuste a tensão FITC do CytomeTer separador Definições na janela de Controlo Inspector de tal modo que o pico da distribuição em forma de sino é inferior a 10 2 de FITC-A. </li><li> Criar um log escalado FSC-A vs. o log FITC-A trama clicando no botão gráfico de pontos na planilha global. Definir um portão triagem R2 no terreno utilizando o botão de porta polígono na barra de ferramenta, de modo que a porta está localizada entre as células + 5% e -5% do centro da distribuição (10% total de células em torno do pico de a de sino curva em forma). </li><li> Colocar num tubo de recolha contendo 1,2 ml de LB contendo 50 ug / ml de ampicilina e 12,5 jig / ml de cloranfenicol na saída do instrumento FACS e classificar 10 6 células que satisfaçam ambos os R1 e R2 portas. </li><li> Retire o tubo de coleta, boné, e gentilmente vortex. </li></ol> 3. metagenômico Enzyme Screening <ol><li> Incubar as células escolhidas (do passo 2.11) a 37 ° C com agitação a 200 rpm até a DO600 atinge 0,5. </li><li> Adicionar 1 ml copiar solução de indução para amplificar o número de cópias fosmid intracelular. Incubam-se as células durante mais 3 horas adicionais a 37 ° C com agitação a 250 rpm. </li><li> Para preparar as células para seleccionar, adicionar 0,5 ml de células em cultura e de um substrato adequado (p-nitrofenil de etilo, p-nitrofenil-β-D-celobiósido, fosfato ou fenilo) para um tubo de 14 ml de fundo redondo numa concentração final de 100 uM. <ol><li> Para uma amostra de controlo, adicionar 0,5 ml de células cultivadas a partir do Passo 3.2 para um tubo de fundo redondo de 14 ml. Adicionar o mesmo volume de PBS como o volume do substrato no passo 3.3. </li></ol></li><li> Incubam-se as duas amostras, a 37 ° C com agitação a 200 rpm durante 3 h. </li><li> Ao mesmo tempo, preparar a máquina de FACS com as mesmas configurações que o passo 2.3. </li><li> Adicionar 5 uL de células (para selecção), e células de controlo para dois tubos de 5 ml de fundo redondo contendo 1 ml de PBS, respectivamente. </li><li> Colocar o tubo contendo células de controle para tele porto de carregamento dos FACS e clique no botão Carregar na Aquisição Painel do software FACS. Ajustar a taxa de eventos para 1.000 - 3.000 eventos / seg, controlando Taxa de Fluxo botões no painel. </li><li> Log criar escalado FSC-A vs. log escalado SSC-Um gráfico de dispersão, clicando no botão gráfico de pontos na planilha global do software e ajustar o portão de dispersão R1 para abranger os eventos singlet (população bacteriana) utilizando o botão Polígono Portão sobre o barra de ferramentas. </li><li> Log criar escalado FSC-A vs. log escalado FITC-Um gráfico de dispersão, clicando no botão gráfico de pontos na planilha e definir uma classificação portão R2 no terreno utilizando o botão Polígono Portão na barra de ferramentas para que menos de 0,1% de células negativas são detectados dentro do portão R2. </li><li> Substitua o tubo de amostra de controlo com o tubo de amostra de triagem, e ajustar a taxa de evento para 1.000 - 3.000 eventos / seg, controlando os botões Taxa de fluxo no painel. </li><li> Coloque um tubo de coleta contendo 0,5ml LB na saída do instrumento FACS e resolver 10 4 células que satisfaçam ambas as portas R1 e R2. Nota: O critério de ordenação pode variar de topo de 0,1% a 5% de FITC-A na porta R2. Neste protocolo, as células top 1% foram recolhidos como positivos. </li><li> Retire o tubo de coleta, boné, e gentilmente vortex. </li><li> Espalhe as amostras de 0,5 ml recolhidos em duas placas de ágar (90 mm de placas de Petri) contendo LB, 50 ug / ml de ampicilina, 12,5 ug / ml de cloranfenicol e Incubar a placa a 37 ° C durante a noite. </li><li> Se necessário, execute rodadas adicionais de triagem para FITC-A enriquecimento, repetindo as etapas 3.1-3.14. </li></ol> 4. Hit Confirmação Seleção e atividade enzimática <ol><li> Citometria de fluxo Análise <ol><li> A partir da placa incubada na Etapa 3.14, selecione uma colônia mostrando ausência de fluorescência usando um seletor de colônia sob a observação de 488 nm de comprimento de onda de um iluminador LED. </li><li> Inocular a colônia selecionado em um 14 ml ro-und fundo do tubo contendo 2 ml de LB contendo 50 ug / ml de ampicilina e 12,5 jig / ml de cloranfenicol a 37 ° C com agitação a 200 rpm até a DO600 atinge 0,5. </li><li> Adicione 2 mL copiar solução de indução para amplificar o número de cópias fosmid intracelular. Incubam-se as células durante mais 3 horas adicionais a 37 ° C com agitação a 250 rpm. </li><li> Preparar um tubo de 14 ml de fundo redondo e adicionar 1 ml de células em cultura (passo 4.1.3). Adicionar um substrato adequado (acetato de p-nitrofenilo, p -nitrophenyl- β -D-celobiósido, ou fosfato de fenilo) a uma concentração final de 100 uM. <ol><li> Para um controlo, adicionar 1 ml das células de cultura do passo 4.1.3 em um tubo de 14 ml de fundo redondo e adicionar o mesmo volume de PBS como o volume do substrato no passo 4.1.4. </li></ol></li><li> Incubam-se as duas amostras, a 37 ° C com agitação a 200 rpm durante 3 h. </li><li> Enquanto isso, prepare a máquina FACS com as mesmas configurações como Passo 2.3. </li><li> Adicionar 5 uL das células de controlo e substrato tratado para tubos de 5 ml de fundo redondo contendo 1 ml de PBS, respectivamente. </li><li> Coloque as células controle tubo contendo no porto de carregamento do dispositivo FACS e clique no botão Carregar na Aquisição Painel do software FACS. Ajustar a taxa de evento para 1.000 - 3.000 eventos / seg usando Vazão botões no painel. </li><li> Clique no botão Aquisição para medir FITC-A. </li><li> Substituir o tubo de amostra de controlo com o tubo de amostra substrato tratado. Ajustar a taxa de evento para 1.000 - 3.000 eventos / seg usando Vazão botões no painel. </li><li> Clique no botão Aquisição para medir FITC-A. </li><li> Comparar a fluorescência dos dois grupos de células através da representação gráfica de um histograma de contagem de células vs o log dimensionado FITC-A. </li></ol></li><li> Outros ensaios para confirmar a actividade da enzima <ol><li> Para posterior identificação dos candidatos de enzimas selecionadas, extrair DNA fosmid usando pro extração padrãoprocedimentos dos kits de extração comerciais e analisar a sequência de nucleotídeos, ou testar a atividade enzimática in vitro 6,7. </li></ol></li></ol>

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The authors have nothing to disclose.

Aumentar a eficiência da produção de biocatalisadores é uma chave para o sucesso de bio-química indústria baseada 9 e metagenome é considerado um dos melhores fonte de enzima natural. Neste sentido, é essencial para o rastreio de novas enzimas a partir da metagenome onde a maioria dos recursos genéticos não têm sido explorados 10. Vários métodos de rastreio foram desenvolvidos que detectam directamente produtos enzimáticos utilizando activadores de transcrição 11, 12, mas...

Uma técnica de ensaio de uma única célula de alto rendimento recentemente desenvolvido permite novas enzimas a ser rastreada a partir de uma biblioteca genética em larga escala com base nas suas actividades funcionais 1. No nível de célula única, as proteínas que regulam a transcrição são empregues para provocar a expressão do gene repórter por detecção pequenas moléculas que são produzidos como um resultado de uma actividade da enzima alvo. Uma abordagem precoce envolvido o isolamento de um operão de fenol-degradantes de Ralstonia eutropha E2 utilizando a expressão genética induzida por substrato rastreio método (SIGEX), em que o substrato induz a expressão de uma proteína repórter 2. NHAr de Pseudomonas putida foi usado para seleccionar benzaldeído desidrogenase 3, e de Corynebacterium glutamicum LysG foi utilizado para o rastreio de alto rendimento de uma nova estirpe de L-lisina-produzir diversas bibliotecas de mutantes 4. Anteriormente, uma enzima screeni genéticasistema ng (GESS) foi proposta como uma plataforma de triagem geralmente aplicável 5. Este sistema utiliza o regulador dimetilfenol-reconhecendo fenol, DMPR, de P. putida. DMPR (E135K), e um mutante de DMPR, também pode ser empregue em GESS (PNP-GESS) para a detecção de p-nitrofenol (PNP). Na presença de enzimas alvo a produção de compostos fenólicos, GESS em E. células de E. coli emite um sinal de fluorescência, permitindo o isolamento rápido de células isoladas usando um classificador de células activadas por fluorescência (FACS). Mas a expressão da enzima metagenômico parece ser mais fraca do que a de enzimas recombinantes convencionais; portanto, GESS foi desenhado para detectar compostos fenólicos com o máximo de sensibilidade ao investigar a combinação de local de ligação ribossómica (RBS) e sequências de terminação junto com ótima condição operacional 5. Uma das vantagens fundamentais do GESS é que este método único permite teoricamente o rastreio de OVer de 200 tipos diferentes de enzimas na base de dados BRENDA (Tabela 1, http: // www.brenda-enzymes.info, 2013.7) simplesmente empregar substratos diferentes. Foi mostrado que a celulase, lipase e hidrolase metil paration (MPH) pode ser detectada utilizando PNP-GESS com substratos apropriados de p-nitrofenilo butirato, p-nitrofenil-cellotrioside, e metil paration, respectivamente 5. Recentemente, provou-se que uma fosfatase alcalina (AP), que é uma das novas enzimas identificadas utilizando PNP-GESS, é o primeiro AP termolábil encontrados em metagenomes marinhos adaptados ao frio 6. Aqui, os detalhes do processo de triagem é apresentado com PNP-GESS detectar as actividades de três tipos diferentes de lipase enzymes-, celulase, e fosfatase alcalina -e rapidamente identificar novas enzimas candidatas a partir de uma biblioteca metagenômico 5,6. Os processos incluem metagenome pré-processamento com PNP-GESS e operação de uma flow citometria classificador. Enquanto os acertos obtidos terão de ser sequenciado para posterior identificação, este protocolo abrange o procedimento acima para os passos de confirmação de actividade enzimática utilizando citometria de fluxo.

<table><tbody><tr><td>CopyControl</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid library production kit&amp;nbsp;</td></tr><tr><td>CopyControl Induction Solution</td><td>Epicentre</td><td>CCIS125</td><td>Fosmid copy induction solution</td></tr><tr><td>EPI300</td><td>Epicentre</td><td>EC300110</td><td>Electrocompetent cell</td></tr><tr><td>pCC1FOS</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid vector</td></tr><tr><td>Gene Pulser Mxcell</td><td>Bio-Rad</td><td></td><td>Electroporation cuvette and electroporate system</td></tr><tr><td>FACSAria III</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry (FACS machine)</td></tr><tr><td>AZ100M</td><td>Nikon</td><td></td><td>Microscope&amp;nbsp;</td></tr><tr><td>UltraSlim&amp;nbsp;</td><td>Maestrogen</td><td></td><td>LED illuminator</td></tr><tr><td>50-ml conical tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>14-ml round-bottom tube&amp;nbsp;</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>5-ml round-bottom tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl phosphate</td><td>Sigma-Aldrich</td><td>N7653</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl &amp;beta;-D-cellobioside</td><td>Sigma-Aldrich</td><td>N5759</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl butylate</td><td>Sigma-Aldrich</td><td>N9876&amp;nbsp;</td><td>Substrate</td></tr><tr><td>Luria-Bertani (LB)</td><td>BD Difco</td><td>244620</td><td>Tryptone 10 g/L, Yeast extract 5 g/L, Sodium Chloride 10 g/L</td></tr><tr><td>Super Optimal broth (SOB)</td><td>BD Difco</td><td>244310</td><td>Tryptone 20 g/L, Yeast extract 5 g/L, Sodium Chloride 0.5 g/L, Magnesium Sulfate 2.4 g/L, Potassium Chloride 186 mg/L</td></tr><tr><td>Super Optimal broth with Catabolite repression (SOC)</td><td></td><td>SOB, 0.4 % glucose</td><td></td></tr><tr><td>2x Yeast Extract Tryptone (2xYT)</td><td>BD Difco</td><td>244020</td><td>Pancreatic digest of Casein 16 g/L, Yeast extract 10 g/L, Yeast extract 5 g/L</td></tr><tr><td>Cell storage media</td><td></td><td></td><td>2x YT broth, 15% Glycerol, 2% Glucose</td></tr><tr><td>pGESS(E135K)</td><td></td><td></td><td>A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator.&lt;br/&gt; See the reference 5 in the manuscript for more details.</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>BD FACSDiva</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry Software Version 7.0</td></tr><tr><td>PBS</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr></tbody></table>

multi-enzyme screening using a high-throughput genetic enzyme screening system

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

Os três substratos fenólicos foram examinados para identificar novas enzimas metagenomic de uma biblioteca metagenome de sedimentos planas oceano-corrente em Taean, Coreia do Sul, seguindo o protocolo proposto. Para a construção da biblioteca, média 30 - sequências metagenome 40 kb foram inseridos no fosmids, que são baseados na E. coli F fator de replicão e apresentados como uma única cópia de uma célula. Note-se que fosmids têm sido amplamente utilizados para a con...

Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzima Triagem Usando um de alta capacidade Enzyme genética Sistema de Triagem

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

multi-enzyme-screening-using-high-throughput-genetic-enzyme-screening

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JoVE Journal

Biology

8.7K visualizações.  Korea Research Institute of Bioscience and Biotechnology. This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate). 

Vídeo: Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

High-throughput screening de enzimas Carboidratos-degradantes Usando Novel Insolúvel substrato cromogénico Kits de Ensaio

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Bioquímica

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicina

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

A tela de elevada capacidade para Biomining Atividade da celulase de Bibliotecas metagenomic

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Biologia

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Alto rendimento Yeast Tela superexpressão Plasmid

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening da atividade endoglucanase de fungos em Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificação de pares Kinase-substrato utilizando High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Genetic Screens

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then assessing the affected organisms for phenotypes of interest. The process can be &#8220;forward&#8221;, where mutations are generated randomly to identify unknown genes responsible for the phenotypes, or it can be &#8220;reverse&#8221;, where specific genes are targeted for mutation to observe what phenotypes are produced.Here, JoVE reviews various types of genetic screens, including those that depend on either loss-of-function or gain-of-function mutations, which respectively decrease or increase the activity of genes. We then explore general protocols for forward and reverse screens in a popular model organism, the nematode worm. Finally, we highlight how screens are applied in research today, for example to better understand gene interactions that may contribute to neurodegenerative diseases.

Forward and Reverse Genetic Screens

Triagens Genéticas

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then ...

Education

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Biologia Avançada

Genética

Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Source: Sch&#252;ckel, J., et al. <a href="https://www.jove.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

Triagem enzimática à base de biomassa cromogênica insolúvel: um ensaio para avaliar a atividade de degradação de enzimas degradadoras de biomassa vegetal

Source: Sch&#252;ckel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. ...