Name: dna-affinity-purified chip (dap-chip) method to determine gene targets for bacterial two component regulatory systems
Uploaded: 2014-07-21T14:00:00
Description: Watch this Scientific Journal Video about DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems at JoVE.com

Vi takker Amy Chen for hendes hjælp i forberedelserne til video shoot og for at demonstrere teknikken. Dette arbejde udføres af ENIGMA: Økosystemer og netværk Integreret med gener og molekylære Forsamlinger (http://enigma.lbl.gov), en videnskabelig Fokusområde Program på Lawrence Berkeley National Laboratory, blev støttet af Ministeriet for Videnskab, Kontoret for biologisk og Environmental Research, af det amerikanske Department of Energy i henhold til kontrakt nr. DE-AC02-05CH11231.

Bemærk: Protokollen nedenfor er skræddersyet til bestemmelse af gen mål for RR DVU3023 fra bakterien Desulfovibrio vulgaris Hildenborough. Det kan tilpasses til en anden transskriptionel regulator af interesse. 1.. Klon og Purify RR <ol><li> Klon RR genet specifikt DVU3023 fra D. vulgaris Hildenborough ind i en Escherichia coli ekspressionsvektor, således at genet er C-terminalt His-mærket og ekspression er under kontrol af en T7-promotor. Bemærk: Fl...

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J., Buck, M., Nixon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+sigma(N)-dependent+promoters+in+bacterial+genomes.">Identification of potential sigma(N)-dependent promoters in bacterial genomes.</a> Microbiology. 146 (12), 3021-3023 (2000).</li><li>Rajeev, L., 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=Systematic+mapping+of+two+component+response+regulators+to+gene+targets+in+a+model+sulfate+reducing+bacterium.">Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium.</a> Genome Biol. 12, 99 (2011).</li><li>Crooks, G. E., Hon, G., Chandonia, J. M., Brenner, S. 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C., 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=Oligomerization+of+the+response+regulator+ComE+from+Streptococcus+mutans+is+affected+by+phosphorylation.">Oligomerization of the response regulator ComE from Streptococcus mutans is affected by phosphorylation.</a> J Bacteriol. 194, 1127-1135 (2012).</li><li>Ladds, J. C., 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=The+response+regulator+Spo0A+from+Bacillus+subtilis+is+efficiently+phosphorylated+in+Escherichia+coli.">The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli.</a> FEMS Microbiol Lett. 223, 153-157 (2003).</li><li>Wen, Y., Feng, J., Scott, D. R., Marcus, E. 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Den her beskrevne DAP-chip-metoden blev med succes anvendt til at bestemme genmål flere RRs i Desulfovibrio vulgaris Hildenborough 7, hvoraf den ene er vist her som et repræsentativt resultat. For RR DVU3023, vælge en kandidat gen mål var ligetil. DVU3025 er placeret umiddelbart nedstrøms for RR-genet og RR-og mål-gener er konserveret i adskillige Desulfovibrio arter, og derudover DVU3025 har en forudsagt sigma54-promotor. EMSA tilvejebringer en enkel metode til hurtigt at teste RR til...

Evnen af ​​bakterier til at overleve og trives er helt afhængig af, hvor godt de er i stand til at opfatte og reagere på forstyrrelser i deres omgivelser, og dette igen er afhængige af deres signaltransduktionssystemer. Antallet af signalsystemer en bakterie koder er blevet kaldt dets &quot;mikrobiel IQ&quot; og kan være en indikation af både variation af miljøet og dets evne til at fornemme flere signaler og finjustere sit svar 1. To komponent signaltransduktion (FKS), er den mest udbredte signalsystemer anvendes af bakterier, og de ​​består af en histidin kinase (HK), som registrerer det eksterne signal og transmitterer via fosforylering til en effektor reaktion regulator (RR) 2. RRs kan have en række af output domæner, og dermed forskellige effektor tilstande, men den mest almindelige reaktion er transkriptionel regulering via et DNA-bindende domæne 1. De signaler, fornemmede og de tilsvarende funktioner i vast størstedelen af ​​fjernsynskamerasystemer fortsat er ukendt. Selv om in vivo-metoder, såsom chip-chip rutinemæssigt anvendes til bestemmelse af genomiske bindingssteder transskriptionsfaktorerne 3, kan de kun anvendes til bakterielle tokomponentsystem RRs hvis aktiverende betingelser eller signaler er kendt. Ofte miljømæssige signaler, der aktiverer en TCS er sværere at afgøre end deres genmål. In vitro microarray assay beskrevet her kan anvendes til effektivt og hurtigt bestemme genmål og forudsige funktioner af de pågældende systemer. Dette assay udnytter det faktum, at RRs kan phosphoryleres og således aktiveres in vitro ved anvendelse småmolekyle donorer som acetylphosphat 4. I denne metode, opkaldt DAP-chip for DNA-affinitetsoprenset-chip (figur 1), er RR-genet af interesse klones med en His-mærke i E. coli, og en efterfølgende oprenset mærkede protein lades bind at klippes genomisk DNA. Proteinet bundet DNA derefter beriget med affinitet-rensning, er det berigede og input DNA forstærkes fluorescensmærkede, lagt sammen og hybridiseret til en flisebelægning array, der er specialfremstillet til organismen af interesse (Figur 1). Microarray eksperimenter er genstand for artefakter og dermed yderligere trin anvendes for at optimere analysen. Et sådant trin er at forsøge at bestemme et mål for RR under undersøgelse under anvendelse af elektroforetisk mobilitet shift assays (EMSA) (se arbejdsgangen i figur 2). Dernæst, efter binding til genomisk DNA og DAP trin, er protein-bundne og input DNA undersøges ved qPCR at se, om den positive mål er beriget i protein-bundne fraktion i forhold til fraktion input, hvilket bekræfter optimale bindingsbetingelser for RR (figur 2). Efter vifte hybridisering, er data analyseres for at finde toppe af højere intensitet signal indikerer genomisk loci hvor proteinet hannonce bundet. Funktioner kan forudsiges for RR baseret på genmål opnået. Target genomiske loci anvendes til at forudsige bindingssted motiver, som derefter eksperimentelt validerede hjælp EMSAs (figur 2). De funktionelle forudsigelser og genmål for RR kan derefter udvides til nært beslægtede arter, som koder for ortologe RRs ved at scanne disse genomer for lignende bindende motiver (figur 2). DAP-chip-metoden kan give et væld af informationer for en FKS, hvor der tidligere var ingen. Fremgangsmåden kan også anvendes til enhver transkriptionel regulator hvis proteinet kan oprenses og DNA-bindingsbetingelser kan bestemmes, og for enhver organisme af interesse med et genom-sekvens til rådighed. <img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig1highres.jpg" width="500" /> Fig. 1. DNA-affinitetsoprenset-chip (DAP-chip) strategi 7.. RR-genet fra organismen af interesse klones med en carboxy-terminal His-mærke i en E. coli-ekspressionsvektoren stamme. Oprenset His-mærket protein aktiveres ved phosphorylering med acetylphosphat, og blandes med klippede genomisk DNA. En portion af bindingsreaktionen gemmes som input DNA, mens resten udsættes for affinitetsoprensning under anvendelse af Ni-NTA-harpiksen. Input og RR-bundet DNA er hele genomet forstærkes og mærket med Cy3 og Cy5, hhv. Det mærkede DNA samles sammen og hybridiseret til en flisebelægning array, som derefter analyseres for at bestemme gen-mål. Figur modificeret og genoptrykt hjælp Creative Commons licens fra 7.. <img alt="Figur 2" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig2highres.jpg" width="500" /> Figur 2.. Resumé af workflow. For enhver renset tagged Protein, begynder ved at bestemme et mål ved hjælp af EMSA. Tillad protein til at binde genomisk DNA og derefter DNA-affinitetsoprensning (DAP) og hele genomet forstærke (WGA) beriget og input DNA. Hvis et gen målet er kendt, anvendes qPCR at sikre, at den kendte mål er beriget med protein-bundne fraktion. Hvis der ingen mål kunne bestemmes, gå direkte til DNA-mærkning og matrix hybridisering. Hvis det ikke kunne observeres berigelse af qPCR, derefter gentage protein-gDNA binding og DAP-WGA trin ved hjælp af forskellige protein beløb. Brug array analyse til at finde toppe og kortlægge dem til at målrette gener. Brug opstrøms regioner af målgener at forudsige bindingssted motiver. Godkend motiverne eksperimentelt ved EMSAs. Brug motivet til at scanne genomer af beslægtede arter, der koder orthologer af RR under studiet, og forudsige gener målrettet på disse arter som godt. Baseret på genmål opnået, kan den fysiologiske funktion af RR og dens orthologer forudsiges. Figur modificeret og genoptrykt ved hjælp af den skabende commons licens fra 7..

<table border="0" cellpadding="0" cellspacing="0" width="719">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Name of Material/Equipment</td>
			<td style="width:248px;">Company</td>
			<td style="width:113px;">Catalog number</td>
			<td style="width:104px;">Comments</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">HisTrapFF column (Ni-Sepharose column)</td>
			<td>GE Lfe Sciences, Pittsburgh, PA, USA</td>
			<td>17-5255-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Akta explorer (FPLC instrument)</td>
			<td colspan="2">GE Lfe Sciences, Pittsburgh, PA, USA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">HiPrep 26/10 Desalting column</td>
			<td>GE Lfe Sciences, Pittsburgh, PA, USA</td>
			<td>17-5087-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Qiaquick Gel extraction kit</td>
			<td>Qiagen Inc, Valencia, CA, USA</td>
			<td align="right">28704</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Biotin-labeled oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">6% polyacrylamide-0.5X TBE precast mini DNA retardation gel</td>
			<td>Life Technologies, Grand Island, NY, USA</td>
			<td>EC63652BOX</td>
			<td>Alternately, you can pour your own gel.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Nylon membrane</td>
			<td>EMD Millipore, Billerica, MA, USA</td>
			<td>INYC00010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Trans-Blot SD Semi-dry electrophoretic transfer cell</td>
			<td>Biorad, Hercules, CA, USA</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Extra thick blot paper, 8 x 13.5 cm</td>
			<td>Biorad, Hercules, CA, USA</td>
			<td>170-3967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">UV crosslinker Model XL-1000</td>
			<td>Fisher Scientific</td>
			<td style="width:113px;">11-992-89</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Nucleic Acid chemiluminescent detection kit (Pierce)</td>
			<td>Thermo fisher Scientific, Rockford, IL, USA</td>
			<td align="right">89880</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Ni-NTA agarose resin</td>
			<td>Qiagen Inc, Valencia, CA, USA</td>
			<td align="right">30210</td>
			<td></td>
		</tr>
		<tr height="119">
			<td height="119" style="height:119px;width:255px;">GenomePlex Whole genome amplification kit (Fragmentation buffer, library preparation buffer, library stabilization solution, library preparation enzyme, 10X amplification master mix, WGA polymerase )</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>WGA2-50RXN</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Nanodrop ND-1000</td>
			<td colspan="2">Thermo Scientific, Wilmington, DE, USA</td>
			<td>For quantitation of DNA</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Perfecta Sybr Green SuperMix, with ROX</td>
			<td>Quanta biosciences</td>
			<td>95055-500</td>
			<td>Any Sybr Green PCR mix may be used</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">PlateMax Ultra clear heat sealing film for qPCR</td>
			<td>Axygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">96 well clear low profile PCR microplate</td>
			<td>Life Technologies, Grand Island, NY, USA</td>
			<td colspan="2">PCR-96-LP-AB-C</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Applied Biosystems StepOne Plus Real time PCR system</td>
			<td>Life Technologies, Grand Island, NY, USA</td>
			<td align="right">4376600</td>
			<td>Any real time PCR system may be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Qiaquick PCR purification kit</td>
			<td>Qiagen Inc, Valencia, CA, USA</td>
			<td align="right">28104</td>
			<td>Any PCR clean up kit may be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Cy3/Cy5-labeled nonamers</td>
			<td>Trilink biotechnologies, San Diego, CA, USA</td>
			<td colspan="2">N46-0001, N46-0002</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Klenow polymerase 50,000U/ml, 3&#39;-5&#39; exo-</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>M0212M</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Hybridization system</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td>This company no longer makes arrays or related items, so alternate sources such as Agilent or Affymetrix will need to be used,</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Custom printed microarrays and mixers</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:255px;">Hybridization kit (2X Hybridization buffer, Hybridization component A, Alignment oligo)</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Wash buffer kit (10X Wash buffer I, II, III, 1 M DTT)</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">GenePix 4200A microarray scanner</td>
			<td colspan="2">Molecular Devices, Sunnyvale CA, USA</td>
			<td>This model has been replaced by superior ones</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">GenePix Pro microarray software</td>
			<td colspan="2">Molecular Devices, Sunnyvale CA, USA</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Nimblescan v.2.4, ChIP-chip analysis software</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

dna-affinity-purified chip (dap-chip) method to determine gene targets for bacterial two component regulatory systems

In vivo-metoder såsom chip-chip er veletablerede teknikker, der anvendes til at bestemme den globale genmål for transkriptionsfaktorer. Men de er af begrænset nytte i at udforske bakterielle to-komponent reguleringssystemer med uncharacterized betingelser aktivering. Sådanne systemer regulerer transkription kun hvis aktiveret i nærvær af unikke signaler. Da disse signaler er ofte ukendt, kan beskrives i denne video artiklen in vitro microarray baserede metode anvendes til at bestemme gen-mål og bindingssteder for respons regulatorer. Dette DNA-affinitetsoprenset chip fremgangsmåde kan anvendes til enhver renset regulator i enhver organisme med et genom. Protokollen indebærer tillader oprenset mærkede protein til at binde til forskudt genomisk DNA og affinitet oprensning protein-bundet DNA, efterfulgt af fluorescerende mærkning af DNA&#39;et og hybridisering til en brugerdefineret flisebelægning array. Foregående trin, der kan anvendes til at optimere analysen for specific regulatorer er også beskrevet. Toppene genereres ved analyse array data anvendes til at forudsige bindingssted motiver, som derefter eksperimentelt validerede. Motivet forudsigelser kan yderligere anvendes til at bestemme genmål af ortologe respons regulatorer på nært beslægtede arter. Vi demonstrerer anvendeligheden af denne metode ved at bestemme genmål og bindingssted motiver og dermed forudsige funktion for en sigma54-afhængig reaktion regulator DVU3023 på miljøområdet bakterie Desulfovibrio vulgaris Hildenborough.

Ovennævnte metode blev anvendt til at bestemme de globale genmål af RRS i modellen sulfat reducerende bakterie Desulfovibrio vulgaris Hildenborough 7. Denne organisme har et stort antal af de pågældende systemer repræsenteret ved over 70 RRs, angiver det brede udvalg af mulige signaler, som den registrerer og reagerer på. In vivo analyser af funktionerne af disse signalsystemer er svære at udføre, da deres signaler og dermed deres aktiverende betingelser er ukendt. Her blev DAP-chip-...

Watch this Scientific Journal Video about DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems at JoVE.com

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

DNA-affinitetsrensede Chip (DAP-chip) metode til at bestemme Gene Mål for bakteriel Tokomponent reguleringsordninger

Denne video artikel beskriver en in vitro microarray baseret metode til at bestemme gen-mål og bindingssteder for to-komponent-system respons regulatorer.

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

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Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

Bacterial Gene Expression Analysis Using Microarrays

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Determining the Contribution of the Energy Systems During Exercise

Bestemmelse af bidrag af Energy Systems under træning

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Brugen af<em&gt; Ex vivo</em&gt; Hele-orgel Imaging og kvantitativ Tissue Histologi at Bestem Bio-distribution af fluorescens-mærkede molekyler

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Brug af en helmonteret immunhistokemisk metode til at studere indervation af galdevejen i<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

The Lactate Dehydrogenase Sequestration Assay ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Laktat Dehydrogenase Sequestration Assay — En simpel og pålidelig metode til at bestemme Bulk Autophagic beslaglæggelse aktivitet i pattedyrceller

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

High-Throughput Live Imaging af mikrokolonier til måling af heterogenitet i vækst og genekspression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Profilering af H3K4me3-modifikation i planter ved hjælp af spaltning under mål og tagmentering

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...