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

The authors would like to thank Dr. Steve Withers and Hong-Ming Chen for providing DNP-Cellobioside substrate.

Before starting this protocol, you will need your metagenomic library stored in a 384 well plate format. In our study, we used the pCC1 copy control fosmid vector in combination with phage T1-resistant TransforMax EPI300-T1R E. coli cells as the library host and stored our plates at -80&deg;C 11.
1. Replication of the Metagenomic Library Plates
<ol>
	<li>Defrost the plates containing your library at 37&deg;C for approximately 20 minutes, or until all wells ...

<ol>
	<li>Rubin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+of+cellulosic+biofuels.">Genomics of cellulosic biofuels.</a> Nature. 454, 841-845 (2008).</li><li>Voget, S., Steele, H. L., Streit, W. R. <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+a+metagenome+derived+halotolerant+cellulase.">Characterization of a metagenome derived halotolerant cellulase.</a> J. Biotechnol. 126, 26-36 (2006).</li><li>Duan, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+partial+characterization+of+novel+genes+encoding+acidic+cellulases+from+metagenomes+of+buffalo+rumens.">Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens.</a> J. Appl. Microbiol. 107, 245-256 (2009).</li><li>Zhang, Y. H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outlook+for+cellulase+improvement:+Screening+and+selection+strategies.">Outlook for cellulase improvement: Screening and selection strategies.</a> Biotech. Advances. 24, 452-481 (2006).</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. And Environ. Microbiol. 43, 777-780 (1982).</li><li>Sharrock, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulase+assay+methods:+a+review.">Cellulase assay methods: a review.</a> J. Biochem and Biophys Methods. 17, 81-106 (1988).</li><li>Kasana, R. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+Insert+Environmental+Genomic+Library+Production.">Large Insert Environmental Genomic Library Production.</a> J Vis Exp. , (2009).</li><li>Martinez, A., Tyson, G. W., 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=Widespread+known+and+novel+phosphonate+utilization+pathways+in+marine+bacteria+revealed+by+functional+screening+and+metagenomic+analyses.">Widespread known and novel phosphonate utilization pathways in marine bacteria revealed by functional screening and metagenomic analyses.</a> Environ Microbiol. 12, 222-238 (2010).</li><li>Wild, J., Hradecna, Z., Szybalski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditionally+amplifiable+BACs:+Switching+from+single-copy+to+high-copy+vectors+and+genomic+clones.">Conditionally amplifiable BACs: Switching from single-copy to high-copy vectors and genomic clones.</a> Genome Research. 12, 1434-1444 (2002).</li><li>Johnson, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sacchirification+of+complex+cellulosic+substrates+by+the+cellulase+system+from+Clostridium+thermocellum.">Sacchirification of complex cellulosic substrates by the cellulase system from Clostridium thermocellum.</a> Appl Environ Microbiology. 43, 1125-1132 (1982).</li><li>Stutzenberger, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulase+Production+by+Thermomonospora+curvata+isolated+from+municipal+solid+waste+compost.">Cellulase Production by Thermomonospora curvata isolated from municipal solid waste compost.</a> Appl Environ Microbiol. 22, 147-152 (1971).</li></ol>

A high throughput screen for the rapid detection of cellulolytic activity from a large insert genomic DNA metagenomic library expressed in E. coli is described in this protocol. This method is an improvement over the CMC/Congo Red assay commonly used in the literature. It is solution based, and allows for one-pot chemistry screening in 384-well plates, with the final output as absorbance readings from a plate reader allowing for quantitative analysis. The automation of each step of this process allows for the un...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

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.

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

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.

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

Research

JoVE Journal

Biology

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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 ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

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 ...

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 ...

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 ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...