Diese Arbeit wurde von der Gordon and Betty Moore Foundation, und durch die UNCF / Merck Science Initiative finanziert.

1. Abschirmblech Vorbereitung <ol><li> Add 25g LB Nährmedium, 15g Agar und 1,5 g Carboxymethylcellulose (CMC) Substrat 1L destilliertes Wasser, und Autoklaven zu sterilisieren. </li><li> Nach dem Autoklavieren können mittel-bis kühl, und fügen Sie geeignete Antibiotika und IPTG zu einer Endkonzentration von 100 &amp;mgr;. </li><li> Gießen Sie 200 ml Medium in je 5 große quadratische Petrischalen / Bioassay Tabletts (240 x 240 x 20mm oder größer). Lassen Platten vollständig trocknen. </li></ol> 2. Endoglucanase Bibliothek Generation und Bildschirm <ol><li> Generieren Sie eine Bibliothek von Endoglucanase katalytische Domäne (n). Dies kann in vielerlei Hinsicht erreicht werden (z. B., site-directed oder zufällige Mutagenese, Rekombination, etc.) und ist durch den Forscher bestimmt. </li><li> Klon der Bibliothek in einen Vektor, so dass sie unter der Kontrolle des T7-Promotors mit dem lac-Operator sind, und enthalten eine pelB Signalsequenz für die Ausrichtung in das Periplasma. Ein Beispiel für einen geeigneten Vektor ist pET22b (+) von Novagen. </li><li> Transformieren Sie die Endoglucanase Bibliothek in E. coli-Stamm BL21 (DE3) und Platte für einzelne Kolonien. </li><li> Inkubieren Transformation über Nacht bei 37 ° C. </li><li> Mit sterilen Zahnstochern, impfen Klone in 96-Well Deep-Well-Platten mit 400μL LB-Medium mit entsprechenden Antibiotika ergänzt. Bei 37 ° C über Nacht unter Schütteln bei 250 Umdrehungen pro Minute. </li><li> Add steriles Glycerin auf 96 gut Nacht-Kulturen bis zu einer Endkonzentration von 10% für die langfristige Lagerung bei -80 ° C. Dies wird die Master-Platte sein. </li><li> Mit einem 96-poligen Stempel, replizieren Platte über Nacht Kulturen auf die Screening-Platten mit IPTG und CMC. Bis zu 5 Sets von Übernacht-Kulturen können auf jeder Vorführung Typenschild geprägt werden. </li><li> Beschriften Sie die Screening-Platten, so dass die Identität und Orientierung der einzelnen 96-Well-Platte bekannt ist. </li><li> Inkubieren Sie die Stempel-Bibliotheken bei Raumtemperatur (17-25 ° C) über Nacht oder bis sichtbare Kolonien gebildet haben. </li><li> Spülen Platte mit destilliertem Wasser, bis alle Spuren von den Kolonien entfernt werden. </li><li> Gießen Kongorot (CR; 0,5% in Wasser) auf gewaschene Platte. Achten Sie darauf, gerade genug, CR hinzufügen, um die Platte zu decken. </li><li> Inkubieren 15 Minuten bei Raumtemperatur. Nicht mehr als 15 Minuten für CR Inkubation. Längere Inkubationszeiten wird in weniger deutliche Halos führen. </li><li> Abgießen CR und fügen Sie eine großzügige Menge (mehr als genug, um die Platte cover) von 1M NaCl. </li><li> Inkubieren 15 Minuten bei Raumtemperatur. </li><li> Abgießen NaCl zu CMC Abbau Halos zu offenbaren. Für eine bessere Auflösung des Halos kann längere Inkubationszeiten mit NaCl oder mehrere NaCl wäscht erforderlich sein. </li><li> Mit einem sterilen Zahnstocher holen aktive Enzyme Klone aus der Master-Platte für die weitere Charakterisierung. </li></ol> 3. Repräsentative Ergebnisse: Ein Beispiel für die Anzeige für diesen hohen Durchsatz-Bildschirm ist in Abbildung 3 dargestellt. Klone können als inaktiv, schwach aktiv, der hochwirksam und von der Größe des Abbaus Zonen identifiziert werden. Dies ist besonders nützlich, wenn ein Enzym mit bekannter Aktivität in der Bibliothek als Referenz enthalten ist. Wie hier gezeigt, ist die Identifizierung von aktiven Enzymen robust und reproduzierbar. Aber bitte beachten Sie, dass eine angemessene Kennzeichnung der Screening-Platten extrem wichtig ist. Der Forscher muss sich aktiv Varianten nach dem Waschen den Kolonien weg zu identifizieren, um sie von der Master-Platte bei -80 ° C für die weitere Charakterisierung gespeichert holen. Schließlich ist es schwierig zu beurteilen, a priori die Beziehung zwischen Aktivitäten auf künstliche vs natürliche Substrate. Deshalb müssen die Forscher testen alle positiven Klone auf industriell relevante Material Protein Fitness zu bestimmen. <img src="/files/ftp_upload/2942/2942fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Schematische Darstellung der Screening-Verfahren. Die Zellen sind mit einer Endoglucanase-Bibliothek transformiert und selektiv für einzelne Kolonien überzogen. Klone gepickt und über Nacht gezüchtet in 96-Well Deep Well Platten für (1) Lagerung bei -80 ° C und (2) Replika-Plattierung auf Platten mit CMC und IPTG zu Endoglucanase Expression zu induzieren. Aktive Klone werden von der Master-Platte bei -80 ° C gelagert abgeholt <img src="/files/ftp_upload/2942/2942fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Klonierung und Expression von Pilz-katalytischen Domänen in E. coli. (A) Endoglucanase Expressionsvektor. Gene wurden in pET22b (+) (Novagen) unter der Kontrolle des T7-Promotor kloniert. (B) Rechts: Endoglucanase-exprimierenden BL21 (DE3) Kolonien. Linkes Bild: CMC Abbau Halos nach dem Waschen und Entwicklung mit Kongorot. <img src="/files/ftp_upload/2942/2942fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Die Ergebnisse der Proben aus CMC / Kongorot Bildschirm. Bilder von Screening-Platten nach dem Waschen und Kongorot Entwicklung genommen. Alle Kolonien, die auf screening Platten wurden von ähnlicher Größe.

<ol>
	<li>Atsumi, S., Hanai, T., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Macrophomina+phaseolina+endoglucanase+(EGL+1)+gene+product+in+bacterial+and+yeast+expression+systems.">Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.</a> Appl Biochem Biotechnol. 81, 153-160 (1999).</li><li>Howard, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+activity+of+a+Phanerochaete+chrysosporium+cellobiohydrolase+(CBHI.1)+expressed+as+a+heterologous+protein+from+Escherichia+coli.">Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli.</a> Afr J Biotechnol. 2, 296-300 (2003).</li><li>Howard, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+chimeric+Phanerochaete+chrysosporium+cellobiohydrolase+expressed+from+Escherichia+coli.">Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli.</a> Afr J Biotechnol. 3, 349-352 (2004).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Appl Env Microbiol. 43, 777-780 (1982).</li><li>Gilkes, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+and+substrate+specificities+of+cellulases+from+cloned+bacterial+genes.">Mode of action and substrate specificities of cellulases from cloned bacterial genes.</a> J Bio Chem. 259, 10455-10459 (1984).</li></ol>

Keine Interessenskonflikte erklärt.

Die hier beschriebene Protokoll ermöglicht eine schnelle und hohen Durchsatz Identifizierung von aktiven Enzymen, mit minimaler Manipulation. Bewegungsmelder ist ziemlich empfindlich und qualitativ spiegelt das Ausmaß der Aktivität innerhalb der Zelle. Seine einfache Bedienung macht diese Methode eignet sich für eine breite Palette von Enzym-Bibliotheken, die nur durch funktionelle Expression in E. begrenzt coli. Darüber hinaus ist Aktivitätsscreening dieser Art nicht zu Pilzcellulasen beschränk...

<table border="1"><tbody><tr><th> Reagens </th><th> Firma </th><th> Produkt-Nummer </th></tr><tr><td> IPTG </td><td> Sigma </td><td> I1284 </td></tr><tr><td> Kongorot </td><td> Sigma </td><td> C6277 </td></tr><tr><td> Carboxymethylcellulose </td><td> Sigma </td><td> 360384 </td></tr><tr><td> NaCl </td><td> Sigma </td><td> S1679 </td></tr><tr><td> Bacto-Agar </td><td> BD Biosciences </td><td> 214030 </td></tr><tr><td> Bioassay-Platten </td><td> Thermo Scientific </td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

Cellulase-Enzyme (Endoglucanasen Cellobiohydrolasen und β-Glucosidasen) hydrolysieren Zellulose in Zucker-Komponente, die wiederum in Kraftstoff Alkohole 1 umgewandelt werden kann. Das Potenzial für die enzymatische Hydrolyse von Zellulose-Biomasse zu erneuerbarer Energie zu versorgen hat sich bemüht, Cellulasen für sparsamen Produktion 2 Ingenieur intensiviert. Von besonderem Interesse sind Pilzcellulasen 3-8, wobei die bereits industriell für Lebensmittel und Textilien verarbeitet wird. Kennzeichnung der aktiven Varianten unter einer Bibliothek von mutierten Cellulasen ist entscheidend für den Engineering-Prozess, aktive Mutanten können weitere für die verbesserten Eigenschaften und / getestet werden oder eine zusätzliche Belastung von Mutagenese. Effizientes Engineering von Pilzcellulasen hat durch einen Mangel an genetischer Werkzeuge für die einheimische Organismen und durch die Schwierigkeiten bei der Expression der Enzyme in heterologen Wirten behindert. Kürzlich entwickelte Morikawa und Mitarbeiter ein Verfahren zur Expression in E. coli der katalytischen Domäne von Endoglucanasen von H. jecorina 3,9, ein wichtiges Industriezentrum Pilz mit der Fähigkeit, Cellulasen in großen Mengen ausscheiden. Functional E. coli Expression wurde auch für Cellulasen aus anderen Pilzen, einschließlich Macrophomina phaseolina 10 und Phanerochaete chrysosporium 11-12 berichtet worden. Wir stellen eine Methode zur Hochdurchsatz-Screening von Pilzen Endoglucanaseaktivität in E. coli. (Abb. 1) Diese Methode verwendet das gemeinsame mikrobielle Farbstoff Kongorot (CR) auf den enzymatischen Abbau der Carboxymethylcellulose (CMC) von wachsenden Zellen auf festem Medium zu visualisieren. Der Aktivitätstest erfordert kostengünstige Reagenzien, minimale Manipulation, und gibt eindeutige Ergebnisse als Zonen des Abbaus (&quot;Halos&quot;) in der Kolonie vor Ort. Obwohl ein quantitatives Maß für enzymatische Aktivität kann mit dieser Methode nicht ermittelt werden, haben wir festgestellt, dass Halo Größe mit insgesamt enzymatische Aktivität in der Zelle korreliert. Die weitere Charakterisierung der einzelnen positiven Klone bestimmt, relative Protein Fitness. Traditionelle bakterielle ganze Zelle CMC / CR Aktivitätstests 13 beinhalten Gießen Agar mit CMC auf Kolonien, die Gegenstand einer Kreuzkontamination oder Inkubation Kulturen in CMC-Agar Brunnen, die weniger für großflächige Experimente ist. Hier berichten wir über ein verbessertes Protokoll, dass die bestehenden Waschverfahren 14 für Cellulase-Aktivität verändert: Zellen, die auf CMC-Agarplatten gezüchtet werden vor CR Färbung entfernt. Unser Protokoll reduziert Kreuzkontamination und ist hoch skalierbar, so dass das schnelle Screening von tausenden von Klonen. Neben H. jecorina Enzyme, haben wir zum Ausdruck gebracht und gesiebt Endoglucanase Varianten aus dem Thermoascus aurantiacus und Penicillium decumbens (siehe Abbildung 2), was darauf hindeutet, dass dieses Protokoll für Enzyme, die aus einer Reihe von Organismen.

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Wir beschreiben eine niedrige Kosten, hohe Durchsatz-Verfahren zum Screening von Pilzen Endoglucanaseaktivität in<em&gt; E. coli.</em&gt; Die Methode beruht auf einer einfachen visuellen Ablesung der Substratabbau, erfordert keine Enzymreinigung und ist hoch skalierbar. Dies ermöglicht das schnelle Screening von großen Bibliotheken der Enzym-Varianten.

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-screening-fungal-endoglucanase-activity-escherichia

Research

JoVE Journal

Biology

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

20.4K Aufrufe.  California Institute of Technology. Wir beschreiben eine niedrige Kosten, hohe Durchsatz-Verfahren zum Screening von Pilzen Endoglucanaseaktivität in E. coli. Die Methode beruht auf einer einfachen visuellen Ablesung der Substratabbau, erfordert keine Enzymreinigung und ist hoch skalierbar. Dies ermöglicht das schnelle Screening von großen Bibliotheken der Enzym-Varianten.

Serie: High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Forschung

Biologie

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 ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-Throughput-Screening und Biosensorik mit Fluorescent C. elegans Die Stämme

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput Screening niedermolekularer Modulatoren von Inward Gleichrichter-Kaliumkanäle

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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 Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Hochdurchsatz-Screening Quantitative Expression und Aufreinigung rekombinanter zu Disulfid-reichen Proteine ​​Venom Produziert in Applied<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Die vielfältigen Vorteile von Protein Co-expression in<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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 ...