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. Preparazione della biblioteca metagenomica con PNP-GESS <ol><li> Costruire una libreria metagenomica in E. coli con un vettore fosmid utilizzando un kit fosmid produzione biblioteca secondo il protocollo 5 del produttore. </li><li> Aliquotare 100 microlitri della libreria per conservazione a -70 ° C, che è una fonte di cellule libreria metagenomiche. Nota: La densità ottica di un campione misurato ad una lunghezza d&#39;onda di 600 nm (OD 600) di questa libreria stock è di circa 100. </li><li> Scongelare 100 ml di dotazione della biblioteca metagenomica sul ghiaccio e inoculare in un pallone da 500 ml contenente 50 ml di Luria-Bertani (LB) e 12,5 mcg / ml cloramfenicolo seguito da 37 ° C di incubazione per 2 ore. </li><li> Raccogliere le cellule in un tubo da 50 ml per centrifugazione a 1.000 xg per 20 min a 4 ° C. </li><li> Risospendere il pellet rapidamente in 50 ml di acqua ghiacciata distillata (DW) e centrifugare a 1000 xg per 20 min a 4 ° C di nuovo. </li><li> resuspend il pellet in 50 microlitri DW ghiacciata con 10% (v / v) di glicerolo. Utilizzare 50 ml di questa aliquota cella per elettroporazione. </li><li> Mettere il composto di cellule electrocompetent 50 pl e pGESS (E135K) 5 DNA (100 ng) in una cuvetta elettroporazione ghiacciata e l&#39;elettroporazione (1,8 kV / cm, 25 uF) la miscela. </li><li> Aggiungere rapidamente 1 ml di brodo eccellente ottimale con cataboliti repressione (SOC) di media e risospendere le cellule delicatamente. </li><li> Trasferire le cellule nel tubo 14 ml a fondo rotondo con una pipetta e incubare a 37 ° C per 1 ora. </li><li> Distribuire 500 ml di cellule recuperate su un LB di 20 x 20 cm Piatto quadrato contenente 12,5 mg / ml cloramfenicolo e 50 mg / ml di ampicillina. Incubare la piastra a 30 ° C per 12 ore. </li><li> Raschiare le colonie utilizzando un raschietto cellulare e raccogliere le cellule in un tubo da 50 ml utilizzando supporti di memorizzazione cella ghiacciata. </li><li> Centrifugare a 1000 xg per 20 min a 4 ° C. Risospendere il pellet in 10 ml di supporti di memorizzazione delle cellule gelideper raggiungere un OD 600 di 100. </li><li> Aliquotare 20 ml di cellule per lo stoccaggio a -70 ° C. </li></ol> 2. Rimozione falsi positivi dalla libreria metagenomiche <ol><li> Scongelare lo stock cellule biblioteca metagenomiche (dal punto 1.13) contenenti metagenomica fosmid DNA e pGESS (E135K) su ghiaccio. </li><li> Seminare 10 ml di cellule in 2 ml LB contenenti 50 ug / ml di ampicillina e 12,5 ug / ml cloramfenicolo in una provetta a fondo rotondo da 14 ml. Incubare a 37 ° C con agitazione a 200 rpm per 4 ore. </li><li> Nel frattempo, accendere la macchina FACS e aprire il software di default FACS. Utilizzare le seguenti impostazioni: diametro della punta dell&#39;ugello, 70 micron; Forward Scatter Area (FSC-A) sensibilità, 300 V-logaritmica di amplificazione; Side Scatter Area (SSC-A) sensibilità, 350 V-logaritmica di amplificazione; Fluoresceina isotiocianato Area (FITC-A) sensibilità, 450 V-logaritmica di amplificazione; parametro di soglia, FSC Un valore 5. Nota: Le impostazioni tipiche sono dateper il dispositivo FACS menzionata nella tabella dei materiali. Regolare le impostazioni per altri dispositivi FACS. </li><li> Diluire le cellule biblioteca metagenomiche dal punto 2.2 con l&#39;aggiunta di 5 ml di campione in una provetta a fondo tondo da 5 ml contenente 1 ml di PBS. </li><li> Mettere il campione biblioteca diluito sul porto di carico dello strumento FACS e fare clic sul pulsante Carica sul Acquisizione Dashboard del software FACS. </li><li> Regolare il tasso di eventi a 1.000 - 1.500 eventi / sec facendo clic e il controllo dei pulsanti di flusso Prezzo sul cruscotto. </li><li> Creare log in scala FSC-A vs log in scala SSC-A grafico a dispersione di un foglio globale facendo clic sul pulsante Plot Dot nella barra degli strumenti. Regolare il cancello dispersione R1 fino a comprendere gli eventi di singoletto (popolazione batterica) utilizzando il pulsante Poligono Porta nella barra degli strumenti. </li><li> Tracciare un istogramma con conta delle cellule contro log in scala FITC-A sul foglio di lavoro facendo clic sul pulsante Istogramma. Poi, regolare la tensione di CIC dal Cytomescheda Impostazioni ter sulla finestra di ispezione controllo tale che il picco della distribuzione a campana è inferiore a 10 2 di FITC-A. </li><li> Creare un registro in scala FSC-A vs. il registro FITC-Una trama cliccando sul tasto Plot Dot sul foglio di lavoro globale. Impostare un ordinamento cancello R2 del terreno con il pulsante Poligono Porta sulla barra degli strumenti in modo che il cancello si trova tra le cellule + 5% e -5% dal centro della distribuzione (cellule totali 10% intorno al picco della campana curva a forma). </li><li> Posizionare un tubo di raccolta contenente 1,2 ml LB contenente 50 ug / ml di ampicillina e 12,5 ug / ml cloramfenicolo all&#39;uscita dello strumento FACS e sort out 10 6 cellule soddisfano sia la R1 e R2 cancelli. </li><li> Rimuovere il tubo di raccolta, berretto, e delicatamente vortice. </li></ol> 3. metagenomiche Enzyme Screening <ol><li> Incubare le cellule ordinati (dal punto 2.11) a 37 ° C con agitazione a 200 giri al minuto fino a quando la OD 600 raggiunge 0,5. </li><li> Aggiungere 1 ml Copy Solution induzione per amplificare il numero di copie fosmid intracellulare. Incubare le cellule per un ulteriore 3 ore a 37 ° C con agitazione a 250 rpm. </li><li> Per preparare cellule selezionare, aggiungere 0,5 ml di cellule coltivate e un substrato (p -nitrophenyl acetato, p -nitrophenyl-β-D-cellobioside, oppure fenile fosfato) in una provetta da 14 ml a fondo rotondo a una concentrazione finale di 100 pM. <ol><li> Per un campione di controllo, aggiungere 0,5 ml di cellule coltivate da Passaggio 3.2 in una provetta a fondo rotondo da 14 ml. Aggiungere lo stesso volume di PBS come il volume del substrato nel passo 3.3. </li></ol></li><li> Incubare le due campioni a 37 ° C con agitazione a 200 rpm per 3 ore. </li><li> Nel frattempo, preparare la macchina FACS con le stesse configurazioni come punto 2.3. </li><li> Aggiungere 5 ml di cellule (per l&#39;ordinamento) e cellule di controllo a due provette da 5 ml a fondo rotondo contenente 1 ml di PBS, rispettivamente. </li><li> Collocare la provetta contenente cellule di controllo Onto tegli caricamento porto delle FACS e fare clic sul pulsante Carica su Acquisizione Dashboard del software FACS. Regolare il tasso di eventi a 1.000 - 3.000 eventi / sec controllando i pulsanti di flusso tasso sul cruscotto. </li><li> Creare log in scala FSC-A vs log in scala SSC-A grafico a dispersione cliccando sul tasto Plot Dot sul foglio di lavoro globale del software e regolare il cancello dispersione R1 fino a comprendere gli eventi singoletto (popolazione batterica) utilizzando il pulsante Poligono porta sul barra degli strumenti. </li><li> Creare log in scala FSC-A vs log in scala coniugati con un grafico a dispersione, cliccando sul pulsante Plot Dot sul foglio di lavoro e impostare un ordinamento porta R2 sulla trama utilizzando il pulsante Poligono Porta sulla barra degli strumenti in modo che meno dello 0,1% di cellule negative vengono rilevati all&#39;interno del cancello R2. </li><li> Sostituire il tubo campione di controllo con il tubo del campione di smistamento, e regolare il tasso di eventi a 1.000 - 3.000 eventi / sec controllando i pulsanti di flusso Prezzo sul cruscotto. </li><li> Posizionare un tubo di raccolta contenente 0,5ml LB all&#39;uscita dello strumento FACS e sort out 10 4 cellule soddisfano entrambe le porte R1 e R2. Nota: Il criterio di ordinamento può variare da cima 0,1% al 5% di FITC-A nel cancello R2. In questo protocollo, top cellule 1% sono state raccolte come positivi. </li><li> Rimuovere il tubo di raccolta, berretto, e delicatamente vortice. </li><li> Distribuire i 0,5 ml campioni raccolti su due piastre di agar (90 mm piatti Petri) contenenti LB, 50 ug / ml di ampicillina e 12,5 ug / ml cloramfenicolo e incubare la piastra a 37 ° C durante la notte. </li><li> Se necessario, eseguire ulteriori cicli di smistamento per FITC-A arricchimento ripetendo i passaggi 3.1-3.14. </li></ol> 4. Hit Selezione e Enzyme Activity Conferma <ol><li> Citometria a flusso <ol><li> Dalla piastra di incubazione a Passo 3.14, selezionare una colonia mostrando alcuna fluorescenza utilizzando un selettore colonia sotto l&#39;osservazione di 488 nm lunghezza d&#39;onda di un illuminatore a LED. </li><li> Inoculare la colonia selezionata in un 14-ml round fondo provetta contenente 2 ml LB contenente 50 ug / ml di ampicillina e 12,5 ug / ml cloramfenicolo a 37 ° C con agitazione a 200 rpm fino alla sua OD 600 raggiunge 0,5. </li><li> Aggiungere 2 ml Copy Solution induzione per amplificare il numero di copie fosmid intracellulare. Incubare le cellule per un ulteriore 3 ore a 37 ° C con agitazione a 250 rpm. </li><li> Preparare un tubo 14 ml a fondo rotondo e aggiungere 1 ml di cellule in coltura (Fase 4.1.3). Aggiungere un substrato (acetato p -nitrophenyl, p -nitrophenyl- β -D-cellobioside, oppure fenile fosfato) ad una concentrazione finale di 100 mM. <ol><li> Per un controllo, aggiungere i 1 ml cellule coltivate dal punto 4.1.3 in un tubo 14 ml a fondo rotondo e aggiungere lo stesso volume di PBS come il volume del substrato nel passaggio 4.1.4. </li></ol></li><li> Incubare le due campioni a 37 ° C con agitazione a 200 rpm per 3 ore. </li><li> Nel frattempo, preparare la macchina FACS con le stesse configurazioni come Passo 2.3. </li><li> Aggiungere 5 ml di cellule di controllo e supporto trattato di provette da 5 ml a fondo rotondo contenente 1 ml di PBS, rispettivamente. </li><li> Mettere le cellule di controllo tubo contenente il porto di carico del dispositivo FACS e fare clic sul pulsante Carica su Acquisizione Dashboard del software FACS. Regolare il tasso di eventi a 1.000 - 3.000 eventi / sec utilizzando i pulsanti di flusso tasso sul cruscotto. </li><li> Fare clic sul pulsante di acquisizione per misurare FITC-A. </li><li> Sostituire il tubo campione di controllo con il tubo del campione substrato trattato. Regolare il tasso di eventi a 1.000 - 3.000 eventi / sec utilizzando i pulsanti di flusso tasso sul cruscotto. </li><li> Fare clic sul pulsante di acquisizione per misurare FITC-A. </li><li> Confrontare la fluorescenza dei due gruppi di cellule tracciando un istogramma del numero di cellule vs. log scalata FITC-A. </li></ol></li><li> Altri test per confermare l&#39;attività enzimatica <ol><li> Per ulteriore identificazione dei candidati enzimi selezionati, estrarre il DNA fosmid usando l&#39;estrazione pro di serieprocedure dei kit di estrazione commerciali e analizzare la sequenza nucleotidica, o testare l&#39;attività enzimatica in vitro 6,7. </li></ol></li></ol>

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The authors have nothing to disclose.

Aumentare l&#39;efficienza della produzione di biocatalizzatori è una chiave per il successo di bio-chimico a base industria 9 e metagenoma è considerato uno dei migliori fonte enzima naturale. In questo senso, è essenziale lo screening di nuovi enzimi dalla metagenome dove la maggioranza delle risorse genetiche non sono state esplorate 10. Diversi metodi di screening sono stati sviluppati che rilevano direttamente i prodotti enzimatici utilizzando attivatori trascrizionali 11, 12 ma ...

Una tecnica saggio cella singola high-throughput recentemente sviluppato permette nuovi enzimi per essere sottoposti a screening da una libreria genetica su larga scala in base alla loro attività funzionali 1. A livello di singola cellula, proteine ​​regolatrici della trascrizione sono impiegati per attivare l&#39;espressione del gene reporter rilevando piccole molecole che sono prodotte come risultato di un&#39;attività enzima bersaglio. Un primo approccio coinvolto l&#39;isolamento di un operone fenolo-degradanti da Ralstonia eutropha E2 utilizzando l&#39;espressione genetica substrato indotta metodo di retinatura (SIGEX), in cui il substrato induce l&#39;espressione di una proteina reporter 2. Nhar di Pseudomonas putida è stato utilizzato per selezionare benzaldeide deidrogenasi 3, e LysG dal Corynebacterium glutamicum è stato utilizzato per lo screening high-throughput di un nuovo ceppo di L-lisina-produzione da diverse librerie di mutanti 4. In precedenza, un enzima screeni geneticaSistema ng (GESS) è stata proposta come una piattaforma di screening generalmente applicabile 5. Questo sistema utilizza il regolatore dimetilfenolo fenolo-riconoscimento, DMPR, di P. putida. DMPR (E135K), e un mutante di DMPR, possono anche essere impiegati in GESS (pNP-GESS) per la rilevazione di p -nitrophenol (pNP). In presenza di enzimi bersaglio producono composti fenolici, GESS in E. coli emette un segnale di fluorescenza, consentendo il rapido isolamento di singole cellule utilizzando un cell sorter fluorescenza attivate (FACS). Ma l&#39;espressione di enzima metagenomico sembra essere più debole di quella degli enzimi ricombinanti convenzionali; Pertanto, GESS è stato progettato per rilevare composti fenolici con la massima sensibilità per indagare la combinazione di ribosomiale sito di legame (RBS) e sequenze di terminazione con ottimali condizioni di funzionamento 5. Uno dei vantaggi fondamentali del GESS è che questo metodo unico permette teoricamente la proiezione di over di 200 diversi tipi di enzimi nel database BRENDA (Tabella 1, http: // www.brenda-enzymes.info, 2013.7) semplicemente utilizzando substrati diversi. E &#39;stato dimostrato che cellulasi, lipasi, e metil parathion idrolasi (MPH) può essere rilevato tramite PNP-GESS con substrati appropriati di p -nitrophenyl butirrato, p -nitrophenyl-cellotrioside, e parathion metile, rispettivamente, 5. Recentemente, è stato dimostrato che una fosfatasi alcalina (AP), che è uno dei nuovi enzimi identificate utilizzando pNP-GESS, è il primo AP termolabile trovato in metagenomi marini adattati freddo 6. Qui, i dettagli del processo di screening è presentato con PNP-GESS rilevare le attività di tre diversi tipi di lipasi enzymes-, cellulasi, e fosfatasi alcalina -e identificare rapidamente nuovi enzimi candidati da una libreria metagenomica 5,6. I processi includono metagenoma pre-elaborazione con PNP-GESS e la gestione di un flOW citometria sorter. Mentre dovranno essere sequenziato per ulteriore identificazione i successi ottenuti, questo protocollo riguarda la procedura fino ai gradini di conferma dell&#39;attività enzimatica citometria a flusso.

<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.

I tre substrati fenolici sono stati esaminati per identificare gli enzimi metagenomiche nuovi da una libreria metagenoma di sedimenti piane oceaniche delle maree a Taean, Corea del Sud, seguendo il protocollo proposto. Per la costruzione della libreria, medi 30 - 40 kb sequenze metagenoma sono stati inseriti fosmidi, che si basano sul E. coli F fattore replicone e presentato come una singola copia in una cella. Si noti che fosmidi sono stati ampiamente utilizzati per la costruzi...

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 screening utilizzando un high-throughput genetica 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 ...

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

Research

JoVE Journal

Biology

8.7K Views.  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). 

Video: 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 degli enzimi carboidrati di degradazione Utilizzando Novel insolubile Chromogenic Substrate Assay Kit

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

Ricerca

Biochimica

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.

Una piattaforma di screening Luciferase basata multiplex per interrogare il cancro-associata trasduzione del segnale in cellule in coltura

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.

Una schermata di High Throughput per Biomining attività Cellulase da librerie metagenomiche

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.

High-throughput iperespressione schermo lievito plasmidi

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.

Throughput Screening elevati di attività Endoglucanase fungine in 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.

High-throughput screening funzionale utilizzando una casalinga 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 ...

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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.

Identificazione delle coppie Kinase-substrato Usando 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

Screening genetici

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

Didattica

JoVE Science Education

Biologia avanzata

Genetica

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.

Screening enzimatico basato sulla biomassa cromogenica insolubile: un test per valutare l'attività di degradazione degli enzimi che degradano la biomassa vegetale

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