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. Vorbereiten der Metagenom-Bibliothek mit pNP-GESS <ol><li> Konstruieren Sie eine Metagenom - Bibliothek in E. coli mit einem Vektor , der eine Fosmid Fosmid Bibliothek Produktions Kit gemäß dem Protokoll des Herstellers 5. </li><li> 100 ul Aliquots der Bibliothek zur Lagerung bei -70 ° C, die eine Quelle der metagenomic Bibliothekszellen ist. Anmerkung: Die optische Dichte eines bei einer Wellenlänge von 600 nm (OD 600) dieser Bibliothek stock gemessenen Probe ist etwa 100. </li><li> Auftauen 100 ul der Vorrats metagenomic Bibliothek auf Eis und Beimpfen für 2 h mit 37 ° C inkubiert in einem 500 ml-Kolben, enthaltend 50 ml Luria-Bertani (LB) und 12,5 &amp; mgr; g / ml Chloramphenicol. </li><li> Ernte der Zellen in einem 50 ml konischen Röhrchen durch Zentrifugation bei 1.000 xg für 20 min bei 4 ° C. </li><li> Das Pellet schnell in 50 ml eiskaltem destilliertem Wasser (DW) und Zentrifuge bei 1000 × g für 20 min bei 4 ° C wieder. </li><li> Resuspend das Pellet in 50 ul eiskaltem DW mit 10% (v / v) Glycerin. Verwenden Sie 50 ul dieser Zelle aliquoten für die Elektroporation. </li><li> Legen Sie die Mischung aus elektro Zellen 50 &amp; mgr; l und pGESS (E135K) 5 DNA (100 ng) in einer eiskalten Elektroporationsküvette und elektroporieren (1,8 kV / cm, 25 &amp; mgr; F) die Mischung. </li><li> Schnell 1 ml SOB-Medium mit Katabolitenrepression (SOC) Medium und die Zellen vorsichtig resuspendieren. </li><li> Übertragen Sie die Zellen in 14 ml Rundbodenrohr mit einer Pipette und Inkubation bei 37 ° C für 1 Stunde. </li><li> Verbreiten 500 ul der gewonnenen Zellen auf LB 20 x 20 cm quadratische Platte, die 12,5 &amp; mgr; g / ml Chloramphenicol und 50 ug / ml Ampicillin. Inkubieren Platte bei 30 ° C für 12 Stunden. </li><li> Schaben die Kolonien eine Zellschaber und sammeln Zellen in einem 50 ml konischen Röhrchen mit eiskalten Zelle Speichermedien. </li><li> Zentrifuge bei 1.000 xg für 20 min bei 4 ° C. Das Pellet in 10 ml eiskaltem Zelle Speichermedieneiner OD 600 von 100 zu erreichen. </li><li> 20 ul Aliquot der Zellen für die Lagerung bei -70 ° C. </li></ol> 2. Entfernen Fehlalarme aus der Metagenom-Bibliothek <ol><li> Auftauen Lager metagenomic Bibliothekszellen (aus Schritt 1.13), die Metagenom-DNA Fosmid und pGESS (E135K) auf Eis. </li><li> Beimpfen von 10 &amp; mgr; l der Zellen in 2 ml LB, enthaltend 50 ug / ml Ampicillin und 12,5 ug / ml Chloramphenicol in einem 14-ml-Rundbodenröhrchen. bei 37 ° C inkubieren, bei 200 rpm für 4 Stunden unter Schütteln. </li><li> Inzwischen schalten Sie die FACS-Maschine und öffnen Sie die Standard-FACS-Software. Verwenden Sie die folgenden Einstellungen: Düsenspitze Durchmesser, 70 &amp; mgr; m; Forward Scatter Area (FSC-A) Empfindlichkeit, 300 V-logarithmische Verstärkung; Side Scatter Area (SSC-A) Empfindlichkeit, 350 V-logarithmische Verstärkung; Fluoresceinisothiocyanat Area (FITC-A) Empfindlichkeit, 450 V-logarithmische Verstärkung; Schwellenparameter, FSC-A-Wert 5. Hinweis: Typische Einstellungen gegeben sindfür die FACS - Gerät in der Tabelle der Materialien erwähnt. Passen Sie die Einstellungen für andere FACS-Geräte. </li><li> Verdünne die metagenomic Bibliothekszellen aus Schritt 2.2 durch Zugabe von 5 ul der Probe auf eine 5-ml-Rundbodenröhrchen mit 1 ml PBS. </li><li> Legen Sie die verdünnte Bibliothek Probe auf die Ladeöffnung des FACS Gerät und klicken Sie auf die Schaltfläche Laden auf dem Acquisition Armaturenbrett des FACS-Software. </li><li> Stellen Sie die Ereignisrate auf 1.000 - 1.500 Veranstaltungen / sec durch Klicken und die Durchflussrate auf dem Armaturenbrett zu steuern. </li><li> Erstellen Sie log skaliert FSC-A vs. Protokoll auf einem globalen Arbeitsblatt SSC-Streudiagramm skaliert durch Klicken auf die Dot Plot - Schaltfläche in der Werkzeugleiste. Stellen Sie die Streu Gate R1 die Singulett-Ereignisse (Bakterienpopulation) mit dem Polygon-Tor-Taste in der Werkzeugleiste zu umfassen. </li><li> Plot ein Histogramm mit Zellzahl gegen log auf dem Arbeitsblatt FITC-A skaliert durch einen Klick auf Histogramm - Taste. Dann stellen Sie die FITC Spannung von der Cytometer Register Einstellungen auf Inspektorfenster steuern, dass die Spitze der glockenförmigen Verteilung von weniger als 10 2 von FITC-A ist. </li><li> Erstellen Sie ein Protokoll skaliert FSC-A vs. das Protokoll FITC-A Plot, indem Sie auf dem globalen Arbeitsblatt auf dem Dot Plot-Schaltfläche klicken. Stellen Sie eine Sortier Tor R2 auf dem Grundstück das Polygon-Gate-Button in der Werkzeugleiste, so dass das Tor zwischen + 5% und -5% Zellen, die aus der Mitte der Verteilung (insgesamt 10% der Zellen rund um den Gipfel des glocken befindet förmige Kurve). </li><li> Legen Sie eine Sammelröhrchen , das 1,2 ml LB sowohl die R1 und R2 Tore befriedigend 50 ug / ml Ampicillin und 12,5 ug / ml Chloramphenicol am Ausgang des FACS Instrument und sortieren 10 6 Zellen enthält. </li><li> Entfernen Sie die Sammelröhrchen, Kappe und sanft Wirbel. </li></ol> 3. metagenomische Enzymscreening <ol><li> Inkubieren der sortierten Zellen (aus Schritt 2.11) bei 37 ° C mit 200 Umdrehungen pro Minute , bis die OD 600 0,5 Schütteln erreicht. </li><li> 1 &amp; mgr; l Induktions Lösung Kopieren Sie die intrazelluläre Fosmid Kopienzahl zu verstärken. Inkubieren der Zellen für weitere 3 h bei 37 ° C mit Schütteln bei 250 Upm. </li><li> Zur Herstellung von Zellen für die Sortierung, 0,5 ml der kultivierten Zellen hinzuzufügen und ein geeignetes Substrat (p - Nitrophenyl Acetat, p - Nitrophenyl-β-D-cellobiosid oder Phenylphosphat) in einen 14-ml - Rundbodenrohr in einer Endkonzentration von 100 uM. <ol><li> Für eine Kontrollprobe mit 0,5 ml der kultivierten Zellen aus Schritt 3.2 in einen 14-ml-Rundbodenrohr. Füge das gleiche Volumen an PBS als das Substratvolumen in Schritt 3.3. </li></ol></li><li> Inkubieren der zwei Proben bei 37 ° C mit bei 200 Upm für 3 h schütteln. </li><li> Inzwischen bereiten die FACS-Maschine mit den gleichen Konfigurationen wie Schritt 2.3. </li><li> Zugabe von 5 &amp; mgr; l der Zellen (für die Sortierung) und Kontrollzellen zu zwei 5 ml-Rundbodenröhrchen mit 1 ml PBS, respectively. </li><li> Das Röhrchen enthält Kontrollzellen auf ter Verladehafen der FACS, und klicken Sie auf die Schaltfläche Laden auf Acquisition Armaturenbrett des FACS-Software. Stellen Sie die Ereignisrate auf 1.000 - 3.000 Veranstaltungen / sec durch auf dem Armaturenbrett Flow Rate Tasten steuern. </li><li> Erstellen Sie log skaliert FSC-A gegen log SSC-A skaliert Streudiagramm mit einem Klick auf Dot Plot - Taste auf dem globalen Arbeitsblatt der Software und stellen Sie die Streu Gate R1 die Singulett - Ereignisse (Bakterienpopulation) mit dem Polygon - Tor - Taste auf das zu umfassen Werkzeugleiste. </li><li> Erstellen Sie log skaliert FSC-A gegen log FITC-A skaliert Streudiagramm , indem Sie auf dem Arbeitsblatt auf dem Dot Plot Schaltfläche klicken und stellen Sie eine Sortier Gate R2 auf dem Grundstück mit dem Polygon - Tor - Taste auf der Werkzeugleiste , so dass weniger als 0,1% negative Zellen sind innerhalb des R2-Gatter erkannt. </li><li> Bringen Sie die Steuerprobenröhrchen mit dem Sortierprobenröhrchen und stellen Sie die Ereignisrate auf 1.000 - 3.000 Veranstaltungen / s durch die Strömungsgeschwindigkeit Tasten am Armaturenbrett steuern. </li><li> Legen Sie eine Sammelröhrchen, das 0,5ml LB am Auslass des FACS - Instrument und aussortieren 10 4 Zellen erfüllt sowohl die R1 und R2 Gattern. Hinweis: Das Sortierkriterium von oben 0,1% bis 5% des FITC-A in der R2 Gate reichen kann. In diesem Protokoll wurden obersten 1% Zellen als positive gesammelt. </li><li> Entfernen Sie die Sammelröhrchen, Kappe und sanft Wirbel. </li><li> Verbreiten Sie die 0,5 ml gesammelten Proben auf zwei Agarplatten (90 mm Petrischalen) mit LB, 50 ug / ml Ampicillin, 12,5 ug / ml Chloramphenicol und Inkubation über Nacht die Platte bei 37 ° C. </li><li> Falls erforderlich, zusätzliche Runden der Sortierung für FITC-A-Anreicherung durch Wiederholen der Schritte 3,1-3,14. </li></ol> 4. Hit Auswahl und Enzymaktivität Bestätigung <ol><li> Durchflusszytometrie-Analyse <ol><li> Von der Platte inkubiert bei Schritt 3.14, wählen Sie eine Kolonie zeigen keine Fluoreszenz eine Kolonie-Picker unter der Beobachtung von 488 nm Wellenlänge einer LED-Beleuchtung verwendet wird. </li><li> Impfen der ausgewählten Kolonie in einem 14-ml-round Boden - Röhrchen mit 2 ml LB unter Schütteln bei 200 rpm 50 &amp; mgr; g / ml Ampicillin und 12,5 ug / ml Chloramphenicol bei 37 ° C , enthaltend bis zu dessen OD 600 0,5 erreicht. </li><li> In 2 ul Induktions Lösung Kopieren Sie die intrazelluläre Fosmid Kopienzahl zu verstärken. Inkubieren der Zellen für weitere 3 h bei 37 ° C mit Schütteln bei 250 Upm. </li><li> Bereiten Sie eine 14 ml-Rundbodenrohr und 1 ml der kultivierten Zellen (Schritt 4.1.3). Hinzufügen eines geeigneten Substrats (p - Nitrophenyl Acetat, p -nitrophenyl- β -D-cellobiosid, oder Phenyl phosphat) in einer Endkonzentration von 100 uM. <ol><li> Zur Kontrolle, fügen Sie die 1 ml kultivierten Zellen aus Schritt 4.1.3 in einem 14 ml-Rundbodenrohr und das gleiche Volumen an PBS als Substrat Volumen in Schritt 4.1.4 hinzuzufügen. </li></ol></li><li> Inkubieren der zwei Proben bei 37 ° C mit bei 200 Upm für 3 h schütteln. </li><li> Inzwischen bereiten die FACS-Maschine mit den gleichen Konfigurationen wie Schritt 2.3. </li><li> Werden 5 ul der Kontrolle und Substrat behandelten Zellen zu 5 ml Rundbodenröhrchen mit 1 ml PBS, respectively. </li><li> Legen Sie die Röhrchen mit Kontrollzellen auf dem Ladeanschluss des FACS-Gerät und klicken Sie auf die Schaltfläche Laden auf Acquisition Armaturenbrett des FACS-Software. Stellen Sie die Ereignisrate auf 1.000 - 3.000 Veranstaltungen / s unter Verwendung von Flow Rate Tasten auf dem Armaturenbrett. </li><li> Klicken Sie auf Acquisition Taste FITC-A zu messen. </li><li> Bringen Sie die Steuerprobenröhrchen mit dem Substrat behandelt Probenröhrchen. Stellen Sie die Ereignisrate auf 1.000 - 3.000 Veranstaltungen / s unter Verwendung von Flow Rate Tasten auf dem Armaturenbrett. </li><li> Klicken Sie auf Acquisition Taste FITC-A zu messen. </li><li> Vergleichen der Fluoreszenz der beiden Gruppen von Zellen durch ein Histogramm der Zellzahl aufgetragen gegen den log FITC-A skaliert. </li></ol></li><li> Andere Tests Enzymaktivität zu bestätigen <ol><li> Zur weiteren Identifizierung der Enzymkandidaten ausgewählt, extrahieren Fosmid DNA-Standard Extraktion Pro mitverfahren aus den handelsüblichen Extraktions Kits und die Nukleotidsequenz zu analysieren, oder das Enzym in vitro Aktivität getestet 6,7. </li></ol></li></ol>

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The authors have nothing to disclose.

Eine Erhöhung der Produktionseffizienz von Biokatalysatoren ist ein Schlüssel für den Erfolg von bio-chemischen Industrie Basis 9 und Metagenom gilt als einer der besten natürlichen Enzymquelle. In diesem Sinne ist es wichtig , neue Enzyme aus der Metagenoms Screening wo die Mehrheit der genetischen Ressourcen 10 nicht erforscht. Mehrere Screening - Verfahren entwickelt worden , die Enzymprodukte unter Verwendung von Transkriptionsaktivatoren 11, 12 , aber diese Techniken erfordern s...

Eine kürzlich entwickelte Hochdurchsatz - Einzelzellen - Assay - Technik ermöglicht neue Enzyme aus einer großen Genbank auf ihre funktionellen Aktivitäten 1 basierend gescreent werden. Auf Einzelzellebene, Transkriptionsproteinen regulieren eingesetzt Expression auszulösen Reportergens durch Abfühlen kleine Moleküle, die als Ergebnis eines Zielenzymaktivität erzeugt werden. Ein früher Ansatz involviert die Isolierung eines Phenol abbau Operon aus Ralstonia eutropha E2 unter Verwendung des substratinduzierte Genexpression Screening (SIGEX) Verfahren, bei dem das Substrat , die Expression eines Reporterproteins 2 induziert. NHAr von Pseudomonas putida wurde verwendet , um Benzaldehyd - Dehydrogenase 3 und lysG aus Corynebacterium glutamicum ausgewählt wurde für die Hochdurchsatz - Screening eines neuen L-Lysin-produzierender Stamm von unterschiedlichen Mutantenbibliotheken 4 verwendet. Zuvor eine genetische Enzym screening - System (GESS) wurde als allgemein anwendbare Screening - Plattform 5 vorgeschlagen. Dieses System verwendet das Phenol-Erkennung Dimethylphenol Regler, DmpR, von P. putida. DmpR (E135K) und eine Mutante von DmpR, können auch in GESS (pNP-GESS) zum Nachweis von p - Nitrophenol (pNP) eingesetzt werden. In Gegenwart von Zielenzymen Herstellung phenolische Verbindungen, GESS in E. coli - Zellen emittiert ein Fluoreszenzsignal, das eine schnelle Trennung von Einzelzellen ermöglicht , eine Fluoreszenz-aktivierten Zellsortierer verwenden (FACS). Aber die Expression des Enzyms metagenomic erscheint als schwächer zu sein, daß der herkömmliche rekombinante Enzyme; Daher wurde GESS ausgelegt phenolische Verbindungen mit maximaler Empfindlichkeit zu erfassen , indem die Kombination der ribosomalen Bindungsstelle (RBS) und Terminatorsequenzen zusammen mit optimalen Betriebszustand 5 untersuchen. Einer der wesentlichen Vorteile von GESS ist, dass diese einzelne Methode ermöglicht es theoretisch das Screening von over als 200 verschiedene Arten von Enzymen in der BRENDA - Datenbank (Tabelle 1, http: // www.brenda-enzymes.info, 2013,7) , indem einfach verschiedene Substrate verwendet. Es zeigte sich, dass Cellulase, Lipase gezeigt, und Methyl - Parathion Hydrolase (MPH) kann mit geeigneten Substraten von p - Nitrophenyl- Butyrat, p - Nitrophenyl-cellotrioside und Methyl - Parathion jeweils 5 unter Verwendung von pNP-GESS nachgewiesen werden. Vor kurzem wurde nachgewiesen , daß eine alkalische Phosphatase (AP), die eine der neuen Enzyme ist pNP-GESS identifiziert verwendet wird , ist das erste thermolabilen AP in kälteangepassten marine Metagenomen gefunden 6. Hier Details des Screening - Prozesses präsentiert mit pNP-GESS die Aktivitäten von drei verschiedenen Arten von enzymes- Lipase Nachweis, Cellulase und alkalische Phosphatase -und schnell neue Kandidaten Enzyme aus einer metagenomic Bibliothek 5,6 identifizieren. Die Verfahren umfassen die Vorverarbeitung Metagenom mit pNP-GESS und Betrieb eines flow-Zytometrie Sortierer. Während die erhaltenen Treffer müssen zur weiteren Identifizierung sequenziert werden, erstreckt sich dieses Protokoll, das Verfahren auf eine Bestätigung zu den Schritten der Enzymaktivität unter Verwendung von Durchflusszytometrie.

<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.

Die drei phenolischen Substrate wurden untersucht, um neue metagenomic Enzyme aus einer Metagenom Bibliothek von Ozean-Wattsedimente in Taean, Südkorea identifizieren durch das vorgeschlagene Protokoll folgen. Für die Bibliothekskonstruktion, durchschnittlich 30 bis 40 kb Metagenoms Sequenzen wurden in Fosmide eingeführt, die auf dem E. beruhen coli F - Faktor Replikon und als eine einzige Kopie in einer Zelle dargestellt. Beachten Sie, dass Fosmide haben zur Konstru...

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).

Screening Multi-Enzym ein High-throughput Genetic Enzymscreening-System verwenden

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 Aufrufe.  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). 

Serie: 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.

Hochdurchsatz-Screening von Kohlenhydrat-abbauenden Enzymen Neuartige Unlöslich chromogenen Assays Kits

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

Forschung

Biochemie

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.

Ein Multiplex-Luciferase-basierte Screening-Plattform zur Abfrage Krebs-assoziierte Signaltransduktion in kultivierten Zellen

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

Medizin

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.

Ein High-Throughput-Screen für Biomining Cellulase Aktivität von Metagenombanken

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

Biologie

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 Hefeplasmid Überexpression Bildschirm

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 von Fungal Endoglucanaseaktivität 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.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Identifikation von Kinase-Substrat-Pairs Mit 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 ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

Hochdurchsatz-Screening für die Protein-basierten Vererbung in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

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

Genetische Tests

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