Name: multi-enzyme screening using a high-throughput genetic enzyme screening system
Uploaded: 2016-08-08T13:00:00
Description: Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.

1. Forberedelse af metagenomisk Bibliotek med pNP-GESS <ol><li> Konstruer en metagenomisk bibliotek i E. coli med en fosmid vektor under anvendelse af en fosmid biblioteksproduktion kit ifølge producentens protokol 5. </li><li> Portion 100 pi af biblioteket til opbevaring ved -70 ° C, hvilket er en kilde til metagenomisk bibliotekets celler. Bemærk: Den optiske densitet af en prøve målt ved en bølgelængde på 600 nm (OD 600) i denne bibliotekets er cirka 100. </li><li> Opt?...

<ol>
	<li>Eggeling, L., Bott, M., Marienhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+screening+methods-biosensors.">Novel screening methods-biosensors.</a> Curr. Opin. Biotech. 35, 30-36 (2015).</li><li>Uchiyama, T., Abe, T., Ikemura, T., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-induced+gene-expression+screening+of+environmental+metagenome+libraries+for+isolation+of+catabolic+genes.">Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes.</a> Nat. Biotechnol. 23 (1), 88-93 (2005).</li><li>Van Sint Fiet, S., van Beilen, J. B., Witholt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+biocatalysts+for+chemical+synthesis.">Selection of biocatalysts for chemical synthesis.</a> Proc. Natl. Acad. Sci. U. S. A. 103 (6), 1693-1698 (2006).</li><li>Binder, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+approach+to+identify+genomic+variants+of+bacterial+metabolite+producers+at+the+single-cell+level.">A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.</a> Genome Biol. 13 (5), R40 (2012).</li><li>Choi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+generalized+and+High-throughput+Enzyme+Screening+System+Based+on+Artificial+Genetic+Circuits.">Toward a generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits.</a> ACS Synthetic Biology. 3 (3), 163-171 (2014).</li><li>Lee, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+psychrophilic+alkaline+phosphatase+from+the+metagenome+of+tidal+flat+sediments.">A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments.</a> BMC Biotechnology. 15 (1), (2015).</li><li>Warnecke, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+and+functional+analysis+of+hindgut+microbiota+of+a+wood-feeding+higher+termite.">Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.</a> Nature. 450 (7169), 560-565 (2007).</li><li>Kim, U. J., Shizuya, H., de Jong, P. J., Birren, B., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+propagation+of+cosmid+sized+human+DNA+inserts+in+an+F+factor+based+vector.">Stable propagation of cosmid sized human DNA inserts in an F factor based vector.</a> Nucleic Acids Research. 20 (5), 1083-1085 (1992).</li><li>Jemli, S., Ayadi-Zouari, D., Hlima, H. B., Bejar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalysts:+application+and+engineering+for+industrial+purposes.">Biocatalysts: application and engineering for industrial purposes.</a> Crc. Cr. Rev. Biotechn. 36 (2), 246-258 (2014).</li><li>Lorenz, P., Eck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomics+and+industrial+applications.">Metagenomics and industrial applications.</a> Nat. Rev. Microbiol. 3 (6), 510-516 (2005).</li><li>Uchiyama, T., Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product-induced+gene+expression,+a+product+responsive+reporter+assay+used+to+screen+metagenomic+libraries+for+enzyme+encoding+genes.">Product-induced gene expression, a product responsive reporter assay used to screen metagenomic libraries for enzyme encoding genes.</a> Applied and environmental microbiology. 76 (21), 7029-7035 (2010).</li><li>Mohn, W. W., Garmendia, J., Galvao, T. C., De Lorenzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+bio-transformations+with+&#224;+la+carte+genetic+traps:+translating+dehydrochlorination+of+lindane+(gamma-hexachlorocyclohexane)+into+lacZ-based+phenotypes.">Surveying bio-transformations with &#224; la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes.</a> Environmental microbiology. 8 (3), 546-555 (2006).</li><li>Tang, S. Y., Cirino, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+a+mevalonate-responsive+regulatory+protein.">Design and application of a mevalonate-responsive regulatory protein.</a> Angew Chem. Int. Ed. 50 (5), 1084-1086 (2011).</li><li>Jha, R. K., Kern, T. L., Fox, D. T., Strauss, C. 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=Engineering+an+Acinetobacter+regulon+for+biosensing+and+high-throughput+enzyme+screening+in+E.+coli+via+flow+cytometry.">Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry.</a> Nucleic Acids Res. 42 (12), 8150-8160 (2014).</li></ol>

Øget produktion effektivitet biokatalysatorer er nøglen til succes for bio-kemiske industri 9 og metagenomet betragtes som en af de bedste naturlige enzym kilde. I denne forbindelse er det vigtigt at screene hidtil ukendte enzymer fra metagenomet hvor størstedelen af de genetiske ressourcer ikke er blevet udforsket 10. Adskillige screeningsmetoder er blevet udviklet som direkte opdage enzymprodukter hjælp transkriptionelle aktivatorer 11, 12, men disse teknikker kræver specifik meta...

En nyligt udviklet high-throughput enkelt-celleassay teknik muliggør hidtil ukendte enzymer, der skal screenes fra en storstilet genetisk bibliotek baseret på deres funktionelle aktiviteter 1. På enkelt celle niveau, er proteiner, der regulerer transkription anvendes til at udløse reportergenekspression ved at registrere små molekyler, der er produceret som et resultat af et mål-enzymaktivitet. En tidlig fremgangsmåde involverede isolering af en phenol-nedbrydende operon fra Ralstonia eutropha E2 anvendelse af substratet-inducerede genetiske ekspression screening (SIGEX) fremgangsmåde, ved hvilken substratet inducerer ekspressionen af et reporter-protein 2. Nhar af Pseudomonas putida blev anvendt til at vælge benzaldehyd dehydrogenase 3, og LysG fra Corynebacterium glutamicum blev anvendt for high-throughput screening af en ny L-lysin-producerende stamme fra forskellige mutantbiblioteker 4. Tidligere en genetisk enzym flotationng-system (GESS) blev foreslået som en generelt gældende screening platform 5. Dette system bruger phenolen-genkendende dimethylphenol regulator, DmpR, af P. putida. DmpR (E135K), og en mutant af DmpR, kan også anvendes i GESS (pNP-GESS) til påvisning af p-nitrophenol (pNP). I nærvær af målenzymer producerer phenolforbindelser, GESS i E. coli celler udsender et fluorescenssignal, giver mulighed for hurtig isolering af enkelte celler under anvendelse af en fluorescens-aktiveret cellesorterer (FACS). Men ekspressionen af ​​metagenomisk enzym synes at være svagere end i konventionelle rekombinante enzymer; derfor blev GESS designet til at detektere phenolforbindelser med maksimal følsomhed ved at undersøge kombinationen af ribosomale bindingssted (RBS) og terminatorsekvenser sammen med optimal driftstilstand 5. Et af de grundlæggende fordele ved GESS er, at denne enkelt metode teoretisk muliggør screening af ovis end 200 forskellige typer af enzymer i BRENDA databasen (tabel 1, http: // www.brenda-enzymes.info, 2013.7) ved blot at ansætte forskellige substrater. Det blev vist, cellulase, lipase, og kan påvises methyl parathion hydrolase (MPH) ved anvendelse pNP-GESS med passende substrater af p-nitrophenyl-butyrat, p-nitrophenyl-cellotrioside og methyl parathion, henholdsvis 5. For nylig blev det vist, at et alkalisk phosphatase (AP), som er en af de hidtil ukendte enzymer identificeret ved anvendelse pNP-GESS, er den første termolabile AP findes i koldadapterede marine metagenomes 6. Her er detaljerne i screeningsprocessen præsenteret med pNP-GESS afsløre aktiviteter tre forskellige typer af enzymes- lipase, cellulase, og alkalisk fosfatase -og hurtigt at identificere nye kandidatlande enzymer fra en metagenomisk bibliotek 5,6. Processerne omfatter metagenomet forbehandling med pNP-GESS og drift af en flow cytometri sorteringsanlæg. Mens de opnåede hits vil skulle sekventeret for yderligere identifikation, denne protokol dækker proceduren indtil trinnene enzymaktivitet bekræftelse ved anvendelse af flowcytometri.

<table border="0" cellpadding="0" cellspacing="0" width="1251">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">CopyControl</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">CCFOS110</td>
			<td style="width:736px;">Fosmid library production kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">CopyControl Induction Solution</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">CCIS125</td>
			<td>Fosmid copy induction solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">EPI300</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">EC300110</td>
			<td>Electrocompetent cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">pCC1FOS</td>
			<td style="width:109px;">Epicentre</td>
			<td style="width:88px;">CCFOS110</td>
			<td>Fosmid vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Gene Pulser Mxcell</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:88px;"></td>
			<td>Electroporation cuvette and electroporate system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">FACSAria III</td>
			<td style="width:109px;">Becton Dickinson</td>
			<td style="width:88px;"></td>
			<td>Flow Cytometry (FACS machine)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">AZ100M</td>
			<td style="width:109px;">Nikon</td>
			<td style="width:88px;"></td>
			<td>Microscope&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">UltraSlim&#160;</td>
			<td style="width:109px;">Maestrogen</td>
			<td style="width:88px;"></td>
			<td>LED illuminator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">50-mL conical tube</td>
			<td style="width:109px;">BD Falcon</td>
			<td style="width:88px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">14-mL round-bottom tube&#160;</td>
			<td style="width:109px;">BD Falcon</td>
			<td style="width:88px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">5-mL round-bottom tube</td>
			<td style="width:109px;">BD Falcon</td>
			<td style="width:88px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">p-nitrophenyl phosphate</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:88px;">N7653</td>
			<td>Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">p-nitrophenyl &#946;-D-cellobioside</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:88px;">N5759</td>
			<td>Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">p-nitrophenyl butylate</td>
			<td style="width:109px;">Sigma-Aldrich</td>
			<td style="width:88px;">N9876&#160;</td>
			<td>Substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Luria- Bertani (LB)</td>
			<td style="width:109px;">BD Difco</td>
			<td style="width:88px;">244620</td>
			<td>Tryptone 10g/L, Yeast extract 5g/L, Sodium Chloride 10g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Super Optimal broth (SOB)</td>
			<td style="width:109px;">BD Difco</td>
			<td style="width:88px;">244310</td>
			<td>Tryptone 20g/L, Yeast extract 5g/L, Sodium Chloride 0.5g/L, Magnesium Sulfate 2.4g/L, Potassium Chloride 186mg/L</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Super Optimal broth with Catabolite repression (SOC)</td>
			<td style="width:88px;"></td>
			<td>SOB, 0.4 % glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Yeast Extract Tryptone (2xYT)</td>
			<td style="width:109px;">BD Difco</td>
			<td style="width:88px;">244020</td>
			<td>Pancreatic digest of Casein 16g/L, Yeast extract 10g/L, Yeast extract 5g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Cell storage media</td>
			<td style="width:109px;"></td>
			<td style="width:88px;"></td>
			<td>2xYT broth, 15 % Glycerol, 2 % Glucose</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:319px;">pGESS(E135K)</td>
			<td style="width:109px;"></td>
			<td style="width:88px;"></td>
			<td style="width:736px;">A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator. 
			See the reference 5 in the manuscript for more details.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Chloramphenicol</td>
			<td style="width:109px;">Sigma</td>
			<td style="width:88px;">C0378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ampicillin</td>
			<td style="width:109px;">Sigma</td>
			<td style="width:88px;">A9518</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">BD FACSDiva</td>
			<td style="width:109px;">Becton Dickinson</td>
			<td style="width:88px;"></td>
			<td>Flow Cytometry Software Version 7.0</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:319px;">PBS</td>
			<td style="width:109px;">Gibco</td>
			<td style="width:88px;">70011-044</td>
			<td>0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4</td>
		</tr>
	</tbody>
</table>

multi-enzyme screening using a high-throughput genetic enzyme screening system

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

De tre phenoliske substrater blev undersøgt for at identificere nye metagenomisk enzymer fra en metagenomet bibliotek af ocean-tidevandsenergi flad sedimenter i Taean, Sydkorea ved at følge den foreslåede protokol. For biblioteket konstruktion, gennemsnitlig 30 - blev 40 kb metagenom sekvenser indsat i fosmids, som er baseret på E. coli F faktor replikon og præsenteres som en enkelt kopi i en celle. Bemærk, at fosmids er ofte blevet brugt til at konstruere komplekse genomi...

Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzym Screening Ved hjælp af en High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Research

JoVE Journal

Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi screening for at identificere Postembryonic fænotyper i C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

High-throughput screening for små-molekyle modulatorer af indadgående Ensretter kaliumkanaler

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biokemisk og High Throughput Mikroskopisk vurdering af Fat Mass i<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

High-throughput Funktionel Screening ved hjælp af en hjemmelavet Dual-glød Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput screening for kemiske modulatorer af post-transkriptionelt regulerede gener

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

Identification of Kinase-substrate Pairs Using High Throughput Screening

Identifikation af kinase-substrat Pairs Brug højkapacitetsscreening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...