Name: a high throughput screen for biomining cellulase activity from metagenomic libraries
Uploaded: 2011-02-01T17:00:00
Description: Watch this Scientific Journal Video about A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries at JoVE.com

Gli autori desiderano ringraziare il Dott. Steve Withers e Hong-Ming Chen per la fornitura di DNP-Cellobioside substrato.

Prima di iniziare questo protocollo, è necessario libreria metagenomica memorizzati in un formato 384 pozzetti. Nel nostro studio, abbiamo usato il pCC1 controllo vettoriale fosmid copia in combinazione con il fago T1-resistente TransforMax EPI300 T1-R E. coli come host biblioteca e conservati i nostri piatti a -80 ° C 11. 1. La replica dei piatti Biblioteca metagenomica <ol><li> Scongelare i piatti contenenti la libreria a 37 ° C per circa 20 minuti, o fi...

<ol>
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C., Salwan, R., Dhar, H., Dutt, S., Gulati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+easy+method+for+detection+of+microbial+cellulases+on+agar+plates+using+Gram's+Iodine.">A rapid and easy method for detection of microbial cellulases on agar plates using Gram's Iodine.</a> Curr. Microbiology. 57, 503-507 (2008).</li><li>Huang, J. S., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+assay+for+cellulase+and+dextranase.">Sensitive assay for cellulase and dextranase.</a> Anal. Biochem. 73, 369-377 (1976).</li><li>Tull, D., Withers, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cellulases+and+xylanases:+A+detailed+kinetic+study+of+the+exo-beta-1,4-glycanase+from+Cellulomonas+fimi.">Mechanisms of cellulases and xylanases: A detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi.</a> Biochemistry. 33, 6363-6370 (1994).</li><li>Martinez, A., Bradley, A. S., Waldbauer, J. R., Summons, R. E., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteorhodopsin+photosystem+gene+expression+enables+photophosphorylation+in+a+heterologous+host.">Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host.</a> PNAS. 104, 5590-5595 (2007).</li><li>Taupp, M., Lee, S., Hawley, A., Yang, J., Hallam, S. 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Uno schermo ad alto rendimento per la rapida individuazione di attività cellulosolitici da una grande libreria genomica metagenomica di DNA espressa in E. coli è descritto in questo protocollo. Questo metodo è un miglioramento rispetto al CMC / Congo saggio Rosso comunemente utilizzati in letteratura. Si tratta di una soluzione basata, e consente di one-pot di screening chimico a 384 pozzetti, con il risultato finale come le letture di assorbanza da un lettore di piastre consentendo analisi quantitativa. L&#...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>qPix2</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>&nbsp;</td>
<td>With 384-pin gridding head</td>
</tr>
<tr>
<td>qFill3</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>&nbsp;</td>
<td>With 384-well manifold</td>
</tr>
<tr>
<td>Varioskan</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RapidStak</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>&nbsp;</td>
<td>Connected to Varioskan</td>
</tr>
<tr>
<td>Micro90 Detergent</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>18100-00</td>
<td>Diluted to 2% in water</td>
</tr>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Major Lab Supplier</td>
<td>&nbsp;</td>
<td>Diluted to 80% in water</td>
</tr>
<tr>
<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0378</td>
<td>12.5mg/mL in ethanol</td>
</tr>
<tr>
<td>LB broth, Miller</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP1426-2</td>
<td>25g/L, autoclaved</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>384-well flat bottom plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3680</td>
<td>&nbsp;</td>
<tr>
<td>L-(+)-Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3256</td>
<td>100mg/mL in water</td>
</tr>
<tr>
<td>Potassium Acetate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P171</td>
<td>50mM in water, autoclaved, adjusted to pH 5.5 with HCl</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP151</td>
<td>&nbsp;</td>
<tr>
<td>Trizma hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T3253</td>
<td>In TE buffer solution, 100mM</td>
</tr>
<tr>
<td>EDTA disodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E5134</td>
<td>In TE buffer solution, 10mM</td>
</tr>
<tr>
<td>2,4-dinitrophenyl cellobioside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Provided by Dr. Steve Withers, UBC</td>
</tr>
<tr>
<td>Dimethyl Sulfoxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D8418</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a high throughput screen for biomining cellulase activity from metagenomic libraries

Cellulosa, la fonte più ricca di carbonio organico del pianeta, ha applicazioni industriali di ampio respiro con enfasi crescente sulla produzione di biocarburanti 1. Metodi chimici per modificare o degradare la cellulosa in genere richiedono acidi forti e temperature elevate. Come tale, i metodi enzimatici sono diventati di primo piano nel processo di bioconversione. Mentre l&#39;individuazione delle cellulasi attivo da ceppi di batteri e funghi è stata in qualche modo efficace, la stragrande maggioranza dei microbi in natura resistere coltura di laboratorio. Genomica ambientale, noto anche come approcci metagenomica, lo screening ha una grande promessa per colmare il divario coltivazione nella ricerca di enzimi bioconversione romanzo. Approcci di screening metagenomica hanno recuperato con successo cellulasi romanzo da ambienti diversi, come i terreni 2, bufali rumine 3 e la termite posteriori-gut 4 con carbossimetilcellulosa (CMC) piastre di agar colorate con colorante rosso congo (basato sul metodo del Teatro e Wood 5). Tuttavia, il metodo CMC è limitata in velocità, non è quantitativo e manifesta un basso rapporto segnale rumore 6. Altri metodi sono stati segnalati 7,8 ma entrambi utilizzare un piatto a base di test di agar, che è indesiderabile per high-throughput screening di grandi biblioteche genomiche inserire. Qui vi presentiamo una soluzione a base di schermo per l&#39;attività cellulasi utilizzando un dinitrofenolo cromogenico (DNP)-cellobioside substrato 9. La nostra biblioteca è stato clonato nel controllo pCC1 copia fosmid per aumentare la sensibilità del test attraverso l&#39;induzione copia numero 10. Il metodo utilizza una pentola chimica in 384 pozzetti con la lettura finale fornito come misura dell&#39;assorbanza. Questa lettura è quantitativa, sensibile e automatizzati con un throughput fino a 100X piastre da 384 pozzetti al giorno, utilizzando un gestore di liquidi e lettore di piastre con annesso sistema di impilaggio.

Watch this Scientific Journal Video about A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries at JoVE.com

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Questo protocollo descrive uno schermo ad alta velocità per l&#39;attività cellulosolitici da una libreria metagenomica espressa in Escherichia coli. Lo schermo è soluzione basata e altamente automatizzata, ed utilizza una pentola chimica nel 384 micropiastre bene con la lettura finale come una misura di assorbanza.

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

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