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

Yazarlar Oynamadı-Cellobioside substrat sağlamak için Dr. Steve Withers ve Hong-Ming Chen teşekkür etmek istiyorum.

Bu protokol başlamadan önce, 384 plaka formatında saklanan metagenomic kütüphane gerekecektir. Bizim çalışmamızda, faj T1 dayanıklı TransforMax EPI300-T1 R E. ile birlikte pCC1 kopyasını fosmid vektör kontrolü kütüphane host ve -80 ° C 11 plakalar saklanır gibi coli hücrelerinde. 1. Metagenomic Kütüphane Tabaklar çoğaltılması <ol><li> Defrost 37 adresinde kütüphane içeren plakalar ° C&#39;de yaklaşık 20 dakika kadar ve...

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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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E. ifade selülolitik aktivitenin büyük bir ekleme genomik DNA metagenomic kütüphane hızlı tespiti için yüksek kapasiteli ekran Bu protokolde coli açıklanmıştır. Bu yöntem, literatürde sıkça kullanılan CMC / Kongo Red tahlil üzerinde bir gelişmedir. Çözüm odaklı ve kantitatif analiz için izin veren bir plaka okuyucu absorbans okumaları son çıkışı ile, 384-kuyucuğu bir pot kimya tarama sağlar. 25 veya daha fazla saat başına 384-kuyu plakaların denetimsiz bir tarama i?...

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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

Selüloz, gezegen üzerinde organik karbon en bol kaynak, biyoyakıt üretimi 1 artan bir vurgu ile geniş çaplı endüstriyel uygulamalar vardır. Selüloz değiştirmek veya aşağılamak için kimyasal yöntemler genellikle güçlü asitler ve yüksek sıcaklıklar gerektirir. Bu nedenle, enzimatik yöntemlerle biyolojik dönüşümünün sürecinde önemli hale gelmiştir. Bakteriyel ve fungal izolatlar aktif sellülazlar belirlenmesi biraz etkili olsa da, doğada mikropların büyük çoğunluğunun laboratuar ekimi karşı. Çevre genomik, metagenomic, tarama yaklaşımlar olarak da bilinen yeni biyolojik dönüşümünün enzimleri için arama ekimi boşluğu dolduruyor büyük umutlar var. Metagenomic tarama yaklaşımları başarıyla topraklar 2, manda rumen 3 ve termit, Kongo kırmızı boya (tiyatrosunun ve Ahşap 5 yöntemi dayalı) ile boyanmış, arka-gut 4 kullanarak karboksimetilselüloz (CMC) agar plakları gibi çeşitli ortamlardan roman sellülazlar iyileşti . Ancak, CMC yöntemi hacmi sınırlı, kantitatif değildir ve düşük sinyal gürültü oranı 6 tezahür. Diğer yöntemler 7,8 bildirilen, ancak her kullanımdan büyük ekleme genomik kütüphaneler yüksek verimli tarama için istenmeyen bir agar plaka bazlı analiz,. Burada selüloz aktivite için bir kromojenik dinitrofenol (DNP) cellobioside substrat 9 kullanarak bir çözüm tabanlı ekran mevcut. Bizim kütüphane ile 10 kopya sayısı indüksiyon testinin duyarlılığı artırmak için fosmid pCC1 fotokopi denetim içine klonlandı. Bu yöntem, son okuma absorbans ölçümü olarak sağlanan 384-iyi mikropleytin bir pot kimya kullanır. Bu okuma kadar bir verim ile bağlı istifleme sistemi ile bir sıvı işleyici ve plaka okuyucu kullanarak günde 100X 384-iyi plakaları, kantitatif, hassas ve otomatik.

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

Bu protokol, Escherichia coli olarak ifade metagenomic kütüphane selülolitik faaliyetleri için yüksek kapasiteli ekran açıklanmıştır. Ekran, çözüm odaklı ve yüksek otomasyonlu ve absorbans ölçümü gibi son okuma ile 384 iyi mikropleytin bir pot kimya kullanır.

Yüksek Verimli Ekran Metagenomic Kütüphaneler selülaz Aktivitesi Biomining

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