Questo lavoro è stato sostenuto da una borsa di studio del Centro per la SLA Packard ricerca presso la Johns Hopkins (ADG), Premio New Innovator un direttore NIH 1DP2OD004417-01 (ADG), NIH R01 NS065317 (ADG), Rita Allen Foundation Scholar Award. ADP è uno studioso Pew nel Scienze Biomediche, sostenuto dal Pew Charitable Trusts.

1. I preparativi per la trasformazione del lievito Questo protocollo è progettato per una decina di piastre a 96 pozzetti, ma può essere scalato verso l&#39;alto o verso il basso di conseguenza. Abbiamo scoperto che questo protocollo non funziona bene per più di venti piastre a 96 pozzetti per ogni turno di trasformazione. La procedura di trasformazione intera (dal punto I.3) dura circa otto ore. <ol><li> Aliquota 5-10μL di DNA plasmidico (100 ng / mL) dalla libreria lievito FLEXGene ORF in ciascun pozzetto di un fondo rotondo a 96 pozzetti con il gestore RapidPlate Biorobot liquido. Luogo scoperto piastre a 96 pozzetti in cappa aspirante da banco pulito durante la notte ad asciugare. Abbiamo trovato efficienze trasformazione simile con il CEN e plasmidi di lievito 2μ. </li><li> Inoculare lievito 200 ml di peptone destrosio (YPD) media (10 g / l estratto di lievito, 20 g / L peptone, 20 g / L di glucosio) con ceppo query in un pallone di Erlenmeyer 500mL sconcertato. Incubare una notte a 30 ° C con agitazione (200 giri). Se necessario, terreni selettivi possono essere utilizzati anche per queste colture starter. </li><li> La mattina seguente, diluire lo sforzo query per OD 600 = 0.1 in 2L di YPD. Agitare per 4-6 ore a 30 ° C (200 rpm) fino a quando la cultura arriva OD 600 = 0,8-1,0. Per una crescita ottimale, crescono due culture 1L in separato 2,8 fiaschi L sconcertato. Se necessario, terreni selettivi può anche essere usato al posto di YPD. </li></ol> 2. Lievito di trasformazione <ol><li> Celle di raccolta per centrifugazione. Riempite quattro tubi monouso 225ml conica con la cultura lievito e far girare a 3000rpm (~ 1800 xg) per 5 minuti in centrifuga da tavolo (Eppendorf 5810R). Eliminare il surnatante e ripetere fino a quando tutte le cellule vengono raccolte. </li><li> Lavare le cellule in 200 ml di distillato in autoclave H 2 O. Risospendere il pellet cellulare agitazione o vortex. Combina le cellule in una provetta conica 225ml. Spin a 3000rpm per 5 minuti. Rimuovere il surnatante. </li><li> Preparare la soluzione 0.1MLiOAc/1XTE (0,1 M LiOAc, 10mM Tris, 1mM EDTA, pH 8 in autoclave acqua distillata). Lavare le cellule in 0.1MLiOAc/1XTE 100 ml. Spin a 3000rpm per 5 minuti. Rimuovere il surnatante. </li><li> Risospendere il pellet cellulare in 70 ml 0,1 M LiOAc/1xTE. Scuotere e / o vortice di assicurare pellet è completamente risospeso. </li><li> Incubare con agitazione a 30 ° C per 30 minuti (200 giri). </li><li> Aggiungi β-mercaptoetanolo per 0,1 M. Incubare con agitazione a 30 ° C per 30 minuti (200 giri). Nota: questo passaggio può essere omesso se si utilizza ceppi di lievito che sono sensibili alle β-mercaptoetanolo. Abbiamo scoperto che questo passaggio non è essenziale per la trasformazione di successo, ma lo fa migliorare l&#39;efficienza di trasformazione. </li><li> Fate bollire 2 ml di DNA sonicato sperma di salmone (10 mg) per 15 minuti, raffreddare e poi su ghiaccio. </li><li> Aggiungere 2 ml di DNA dello sperma di salmone bollito alla miscela di cellule. Attenzione: può formare un precipitato dopo l&#39;aggiunta del DNA dello sperma di salmone alla miscela di cellule. Utilizzando un Pipetman e 200 microlitri punta, rimuovere con attenzione l&#39;eventuale precipitato che si forma, perché questo potrebbe interferire con pipettaggio a valle. </li><li> 50μL aliquota di miscela di cellule in ciascun pozzetto della piastra a 96 pozzetti utilizzando il Rapidplate BioRobot (può anche usare pipetta multicanale). Non mescolare. </li><li> Incubare a temperatura ambiente per 30 minuti senza agitare. </li><li> Preparare 200 ml di 40% PEG-3350/10% DMSO/0.1M LiOAc (160ml del 50% PEG-3350, DMSO 20 mL, 20 mL LiOAc 1M). Preparare la soluzione immediatamente prima dell&#39;uso. </li><li> Aggiungere 125μL di PEG / LioAc / DMSO soluzione a ciascun pozzetto. Mix di aspirare e dispensare sospensione cellulare otto volte con RapidPlate. </li><li> Incubare a temperatura ambiente per 30 minuti senza agitare. </li><li> Scossa di calore le cellule a 42 ° C per 15 minuti in un incubatore a secco. Non impilare piatti. Nota: Abbiamo trovato che questo passo shock termico non è essenziale per la trasformazione di successo. Per alcuni ceppi di lievito (per esempio, i mutanti sensibili alla temperatura), shock termico è deleteria. Abbiamo già ridotto il tempo shock termico a un minuto e sono stati in grado di trasformare con successo un ceppo di lievito portatori di una mutazione sensibile ypt1 temperatura con questo protocollo. </li><li> Spin piastre a 2500rpm (ca. 1100 xg) per 5 minuti, utilizzando adattatori centrifuga per accogliere piastre da 96 pozzetti. Rimuovere la soluzione PEG da piastre invertendo oltre secchio dei rifiuti. Blot piastra capovolta su carta assorbente per eliminare il liquido residuo (cellule resterà sul fondo dei pozzi). </li><li> Lavare le cellule con l&#39;aggiunta di 200μL di terreno minimo (ad esempio, SD /-Ura) in ciascun pozzetto con RapidPlate. </li><li> Spin piastre a 2500rpm per 5 minuti e rimuovere sopranatante invertendo secchio dei rifiuti più e assorbente su carta assorbente. </li><li> Aggiungere 200μL di terreno minimo (ad esempio, SD /-Ura) in ciascun pozzetto con RapidPlate. </li><li> Incubare a 30 ° C per 2 giorni senza agitare. Dopo 2 giorni, piccole colonie di cellule dovrebbero iniziare a modulo in fondo di ogni successo trasformato bene. </li></ol> 3. Spotting test <ol><li> Dispensare 200μL di raffinosio mezzi di comunicazione basati minimo (ad esempio SRaf /-Ura) in ciascun pozzetto diun nuovo fondo piatto piastra da 96 pozzetti. </li><li> Mescolare ciascun pozzetto della piastra di trasformazione da aspirare e dispensare sospensione cellulare otto volte con Rapidplate. </li><li> Inoculare le piastre con SRaf 5μL di miscela di cellule dalla piastra di trasformazione. </li><li> Incubare a 30 ° C per un giorno. Piccole colonie dovrebbero essere presenti nella parte inferiore di ogni bene dopo un giorno. </li><li> Colonie Spot su SD /-Ura e SGal /-Ura piastre in cappuccio. Sterilizzare 96-bullone replicatore (Frogger) mediante esposizione alla fiamma. Mescolare colonie con Frogger prima spotting su piastre selettive dei media. Dopo spotting, permettono piastre ad asciugare cappuccio per 5-10 minuti. </li><li> Incubare a 30 ° C per 2-3 giorni. </li></ol> Piastre di fotografia con fotocamera digitale, e confrontare visivamente la crescita di colonie su SGal /-Ura piastre di trovare colonie in cui tossicità ceppo query è migliorato (una crescita più lenta / colonie meno densa) o soppressa (la crescita più veloce / più dense colonie). 4. Rappresentante dei risultati: <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="Figura 1"/> Figura 1. Sovraespressione schermo lievito plasmide per identificare soppressori ed esaltatori di TDP-43 tossicità. TDP-43 è una proteina umana che è stato implicato nella patogenesi della sclerosi laterale amiotrofica (malattia di Lou Gehrig). Aggregati citoplasmatici di TDP-43 si accumulano nel cervello e neuroni del midollo spinale di pazienti affetti da SLA 17. Esprimendo TDP-43 nei risultati lievito cellule aggregazione e citotossicità 18. Abbiamo utilizzato questo sistema modello per definire i meccanismi di TDP-43 tossicità 19,20. Presenti sono degli esempi rappresentativo di piatti dal nostro lievito TDP-43 schermo modificatore di tossicità. Queste lastre visualizzare colonie con un sistema integrato di galattosio-inducibile TDP-43 plasmide e anche trasformato con i plasmidi della libreria FLEXGene espressione ORF. La piastra a sinistra contiene glucosio, che reprime l&#39;espressione di TDP-43 o il plasmidi FLEXGene. La piastra di destra contiene galattosio, che induce l&#39;espressione di TDP-43 e la ORF in plasmidi FLEXGene. La freccia verde indica una colonia trasformato con un plasmide che sopprime la tossicità di TDP-43. La freccia rossa indica una colonia trasformato con un plasmide che esalta la tossicità di TDP-43.

<ol>
	<li>Nurse, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nobel+Prize+and+beyond:+an+interview+with+Sir+Paul+Nurse.+Interview+by+Susan+R.+Owens.">The Nobel Prize and beyond: an interview with Sir Paul Nurse. Interview by Susan R. Owens.</a> EMBO Rep. 3, 204-206 (2002).</li><li>Hartwell, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nobel+Lecture.+Yeast+and+cancer.">Nobel Lecture. Yeast and cancer.</a> Biosci Rep. 22, 373-394 (2002).</li><li>Stevens, T., Esmon, B., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+in+the+yeast+secretory+pathway+are+required+for+transport+of+carboxypeptidase+Y+to+the+vacuole.">Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole.</a> Cell. 30, 439-448 (1982).</li><li>Novick, P., Ferro, S., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+of+events+in+the+yeast+secretory+pathway.">Order of events in the yeast secretory pathway.</a> Cell. 25, 461-469 (1981).</li><li>Novick, P., Field, C., Schekman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+23+complementation+groups+required+for+post-translational+events+in+the+yeast+secretory+pathway.">Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway.</a> Cell. 21, 205-215 (1980).</li><li>Sopko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+pathways+and+phenotypes+by+systematic+gene+overexpression.">Mapping pathways and phenotypes by systematic gene overexpression.</a> Mol Cell. 21, 319-330 (2006).</li><li>Alberti, S., Gitler, A. D., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+Gateway((R))+cloning+vectors+for+high-throughput+genetic+analysis+in+Saccharomyces+cerevisiae.">A suite of Gateway((R)) cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.</a> Yeast. 24, 913-919 (2007).</li><li>Gelperin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+genetic+analysis+of+the+yeast+proteome+with+a+movable+ORF+collection.">Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.</a> Genes Dev. 19, 2816-2826 (2005).</li><li>Hu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaching+a+complete+repository+of+sequence-verified+protein-encoding+clones+for+Saccharomyces+cerevisiae.">Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.</a> Genome Res. 17, 536-543 (2007).</li><li>Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+profiling+of+the+Saccharomyces+cerevisiae+genome.">Functional profiling of the Saccharomyces cerevisiae genome.</a> Nature. 418, 387-391 (2002).</li><li>Boone, C., Bussey, H., Andrews, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+genetic+interactions+and+networks+with+yeast.">Exploring genetic interactions and networks with yeast.</a> Nat Rev Genet. 8, 437-449 (2007).</li><li>Mnaimneh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+essential+gene+functions+via+titratable+promoter+alleles.">Exploration of essential gene functions via titratable promoter alleles.</a> Cell. 118, 31-44 (2004).</li><li>Parsons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat Biotechnol. 22, 62-69 (2004).</li><li>Schuldiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+the+function+and+organization+of+the+yeast+early+secretory+pathway+through+an+epistatic+miniarray+profile.">Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.</a> Cell. 123, 507-519 (2005).</li><li>Tong, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+genetic+analysis+with+ordered+arrays+of+yeast+deletion+mutants.">Systematic genetic analysis with ordered arrays of yeast deletion mutants.</a> Science. 294, 2364-2368 (2001).</li><li>Tong, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+mapping+of+the+yeast+genetic+interaction+network.">Global mapping of the yeast genetic interaction network.</a> Science. 303, 808-813 (2004).</li><li>Neumann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitinated+TDP-43+in+frontotemporal+lobar+degeneration+and+amyotrophic+lateral+sclerosis.">Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.</a> Science. 314, 130-133 (2006).</li><li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proc Natl Acad Sci U S A. 105, 6439-6444 (2008).</li><li>Elden, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS.">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466, 1069-1075 (2010).</li><li>Johnson, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+is+intrinsically+aggregation-prone,+and+amyotrophic+lateral+sclerosis-linked+mutations+accelerate+aggregation+and+increase+toxicity.">TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity.</a> J Biol Chem. 284, 20329-20339 (2009).</li><li>Cooper, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beer+and+Bread+to+Brains+and+Beyond:+Can+Yeast+Cells+Teach+Us+about+Neurodegenerative+Disease?.">Beer and Bread to Brains and Beyond: Can Yeast Cells Teach Us about Neurodegenerative Disease?.</a> Neurosignals. 16, 52-62 (2008).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+is+part+of+a+diverse+and+highly+conserved+interaction+network+that+includes+PARK9+and+manganese+toxicity.">Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.</a> Nat Genet. 41, 308-315 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Qui vi presentiamo un protocollo per eseguire un elevato throughput schermo plasmide sovraespressione nel lievito. Questo approccio permette di screening rapido e imparziale per modificatori genetici di molti diversi fenotipi cellulari. Usando questo approccio, un ricercatore può schermo una porzione significativa del genoma del lievito in una questione di settimane. Questo approccio imparziale consente inoltre l&#39;identificazione di modifica, che non possono essere previste in base ai risultati precedenti. Abbiamo u...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> BioRobot RapidPlate </td><td> Qiagen </td><td> 9000490 </td></tr><tr><td> 96 bullone replicatore (Frogger) </td><td> V &amp; P scientifico </td><td> VP404 </td></tr><tr><td> FLEXGene ORF Biblioteca </td><td> Istituto di Proteomica, Harvard Medical School </td><td></td></tr><tr><td> Centrifuga da tavolo </td><td> Eppendorf </td><td> 5810R </td></tr><tr><td> 500mL fiasco sconcertato </td><td> Bellco </td><td> 2543-00500 </td></tr><tr><td> 2.8L triplo sconcertato Fernbach fiasco </td><td> Bellco </td><td> 2551-02800 </td></tr><tr><td> 100μL Rapidplate puntali </td><td> Axygen </td><td> ZT-100-RS </td></tr><tr><td> 200μL Rapidplate puntali </td><td> Axygen </td><td> ZT-200-RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

Il lievito, Saccharomyces cerevisiae, è un sistema potente modello per la definizione di meccanismi fondamentali di molti importanti processi cellulari, tra cui quelle con rilevanza diretta per la malattia umana. A causa della sua breve tempo di generazione e ben caratterizzati genoma, uno dei principali vantaggi sperimentale del sistema modello lievito è la capacità di eseguire schermi genetico per identificare i geni e le vie che sono coinvolti in un dato processo. Nel corso degli ultimi 30 anni tale schermi genetici sono stati utilizzati per spiegare il ciclo cellulare, via secretoria, e molti altri aspetti altamente conservato della biologia delle cellule eucariotiche 1-5. Negli ultimi anni, diverse biblioteche genoma dei ceppi di lievito e plasmidi sono stati generati 6-10. Queste raccolte permettono ora per l&#39;interrogatorio sistematico della funzione del gene utilizzando guadagno e la perdita di funzione approcci 11-16. Qui forniamo un protocollo dettagliato per l&#39;utilizzo di un high-throughput protocollo di lievito di trasformazione con un robot di gestione dei liquidi per eseguire uno schermo plasmide iperespressione, utilizzando una libreria di 5.500 schierati plasmidi lievito. Abbiamo utilizzato questi schermi per identificare i modificatori genetici di tossicità associata con l&#39;accumulo di proteine ​​umane di aggregazione a rischio di malattie neurodegenerative. I metodi presentati qui sono facilmente adattabili allo studio di altri fenotipi cellulari di interesse.

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

Qui si descrive uno schermo plasmide sovraespressione in<em&gt; Saccharomyces cerevisiae</em&gt;, Utilizzando una libreria schierate plasmide e un elevato throughput protocollo di trasformazione di lievito con un robot di manipolazione dei liquidi.

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-yeast-plasmid-overexpression-screen

Research

JoVE Journal

Biology

16.3K Views.  University of Pennsylvania School of Medicine. Qui si descrive uno schermo plasmide sovraespressione in Saccharomyces cerevisiae, Utilizzando una libreria schierate plasmide e un elevato throughput protocollo di trasformazione di lievito con un robot di manipolazione dei liquidi.

Video: High-throughput Yeast Plasmid Overexpression Screen

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 ...

Ricerca

Biologia

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

High-throughput per l&#39;analisi Saccarificazione materiali lignocellulosici

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microiniezioni di Sea Urchin zigoti

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

High-throughput Titolazione della luciferasi che esprimono ricombinanti virus

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manuale Schermo piccole molecole si avvicina ad alta velocità Uso embrioni di zebrafish

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput<em&gt; Danio Rerio</em&gt; Energia spese Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...