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

저자는 DNP - Cellobioside 기판을 제공하는 스티브 위더스 및 홍콩 - 밍 첸 감사하고 싶습니다.

이 프로토콜을 시작하기 전에, 당신은 384 잘 플레이트 형식으로 저장하여 metagenomic 라이브러리가 필요합니다. 우리의 연구에서는, 우리는 파지 T1 방지 TransforMax EPI300 - T1 R E.와 함께 pCC1 복사 제어 fosmid 벡터를 사용 도서관 호스트 및 -80 ° C 11 시에 우리 접시 저장된 대장균 세포. 1. Metagenomic 도서관 플레이트의 복제 <ol><li> 37 라이브러리를 포함?...

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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. 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대형 삽입 게놈의 DNA의 metagenomic 라이브러리에서 cellulolytic 활동의 신속한 검출을위한 높은 처리량 화면 E.으로 표현 대장균이 프로토콜에 설명되어 있습니다. 이 방법은 일반적으로 문학에 사용되는 CMC / 콩고 레드 분석을 통해 개선이다. 이 솔루션을 기반으로하고, 정량 분석​​을 위해 허용하는 플레이트 판독기에서 흡광도 수치로 최종 출력으로 384 잘 접시에 한 냄비 화학 검사 ?...

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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

셀룰로스, 지구상의 유기 탄소의 가장 풍부한 원천은, 생물 연료 생산 1 증가 중심으로 광범위한 산업 애플 리케이션을했다. 셀룰로스를 수정하거나 저하에 화학 방법은 일반적으로 강한 지방산과 높은 온도를 필요로합니다. 따라서, 효소 방법은 bioconversion 과정에서 두드러진되고 있습니다. 박테리아와 곰팡이 격리에서 Active cellulases의 식별은 다소 효과가있다지만, 자연 미생물의 대부분은 실험 재배에 저항. 또한 metagenomic, 심사 방식으로 알려진 환경 게놈은 소설 bioconversion 효소에 대한 검색 재배 격차를 브리징 큰 약속을했습니다. 토양 2, 버팔로 제일위 3 콩고 레드 염료 (테더와 우드 5 방법에 따라) 물들일 흰개미 뒷다리 - 직감 4 사용 carboxymethylcellulose (CMC) 한천 플레이트와 같은 다양한으로 Metagenomic 심사 방식이 성공적으로 환경에서 소설 cellulases를 발견했습니다. 그러나, CMC 메서드가 처리량 제한됩니다, 양적되지 않고 잡음 비율 6 낮은 신호를 승객 명단. 다른 방법은 7,8를보고 있지만 각 사용 대형 삽입 게놈 라이브러리의 높은 처리량 검사 바람직하지는 한천 플레이트 기반의 분석.했습니다 여기서 우리는 솔루션을 기반 chromogenic dinitrophenol (DNP) cellobioside 기판 9를 사용 cellulase 활동에 대한 화면을 제시한다. 우리 도서관은 복사 번호 유도 열을 통해 분석 감도를 높이기 위해 fosmid pCC1 복사 제어에 복제되었습니다. 방법은 흡광도 측정으로 제공되는 최종 판독과 함께 384 잘 microplates에 한 냄비 화학을 사용합니다. 이 저장되어 첨부 스태킹 시스템으로 액체 처리기 및 플레이트 판독기를 사용하여 하루에 100X 384 잘 접시 최대의 처리량과 양적 민감하고 자동으로 진행됩니다.

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

이 프로토콜은 대장균의 표현 metagenomic 도서관에서 cellulolytic 활동에 높은 처리량 화면을 설명합니다. 화면이 솔루션을 기반으로하고 고도의 자​​동화, 그리고 흡광도 측정으로 최종 판독과 384 잘 microplates에 한 냄비 화학을 사용합니다.

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

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

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JoVE Journal

Biology

Large-Scale Screens of Metagenomic Libraries

Metagenomic 도서관 대규모 화면

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

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높은 처리량 효모 플라스미드 Overexpression 화면

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High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균</em

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

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10 나노 그램 총 RNA의 단일 읽기와 결합하여 최종 mRNA - Seq의 Illumina 도서관

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

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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

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

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

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

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

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후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

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

높은 처리량 시퀀싱을 활용하여 전체 - 단백질 포화 돌연변이 유발 도서관의 기능 평가를위한 프로토콜

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