Name: electrochemical detection of deuterium kinetic isotope effect on extracellular electron transport in shewanella oneidensis mr-1
Uploaded: 2018-04-16T14:30:00
Description: Watch this Scientific Journal Video about Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1 at JoVE.com

Dette arbeidet ble økonomisk støttet av en Grant-in-Aid for spesielt forfremmet forskning fra Japan Society for fremme av vitenskap (JSPER) KAKENHI bevilgning nummer 24000010, 17H 04969, og JP17J02602, oss Office av Naval Research globalt (N62909-17-1-2038). Y.T. er stipendiat JSP og støttes av JSP gjennom programmet for ledende utdannet skoler (fortjeneste).

1. dannelsen av en Monolayer Biofilm S. oneidensis MR-1 på en ITO elektrode (figur 1)
Merk: For å hindre forurensning av elektrokjemiske reaktoren med andre mikrober, alle medier, redskaper, og komponentene i elektrokjemiske reaktoren skal steriliseres på forhånd. Når benytter S. oneidensis MR-1 celler og konstruere de elektrokjemiske reaktorene, alle prosedyrer bør gjennomføres på en ren benk.
<ol><li>Dyrking av S. oneidensis ...

<ol>
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Våre hele-celle elektrokjemiske analysen har flere tekniske fordeler sammenlignet med protein elektrokjemi. Mens protein rensing krever flere trinn tidkrevende prosedyrer, tar våre hele-cellen metoden en dag av Self-organisert biofilm formasjon etter cellekultur. For å oppnå en stabil samhandling mellom OM c- Cyts og elektroden, vi må bare sterilisering og rengjøring av elektroden overflaten; det krever ikke elektrode endringer for organisering retningen på proteiner4, f.eks</e...

Elektrokjemiske teknikker som direkte kjennetegner en redoks protein i en intakt bakteriell celle har nylig dukket opp siden oppdagelsen av metall-reduserende mikrobiell belastninger, S. oneidensis MR-1 eller Geobacter sulfurreducens PCA, som har ytre membran c-type cytochrome komplekser (OM c-Cyts) utsatt for cellen utvendig1,2,3,4,5. OM c- Cyts formidle elektronet transport fra åndedretts kjeden til fast underlag ligger extracellularly. Denne transporten kalles ekstracellulære elektronet transport (lunsj)1,6 og er en kritisk prosess for nye bioteknologi, for eksempel mikrobielle brenselceller6. Derfor, for å forstå de underliggende EET kinetics og mekanismer, og koblingen til mikrobiell fysiologi, OM c -Cyts er gransket bruker hele-celle elektrokjemi4,7, kombinert med mikroskopi 8 , 9, spektroskopi10,11og molekylærbiologi2,4. I kontrast er metoder for å undersøke virkningen av lunsj-assosiert kasjon transport, f.eks protoner, på EET kinetics i levende celler neppe etablert, til tross for proton transport over bakteriell membraner har en avgjørende rolle i signalering, homeostase og energi produksjon12,13,14. I denne studien, vi beskriver en teknikk for å undersøke virkningen av proton transport EET kinetics i S. oneidensis MR-1 cellen med hele celle elektrokjemiske mål, som krever identifikasjonen av trinnet hastighetsbegrensning i mikrobiell gjeldende produksjon15.
En direkte måte å vurdere bidrag av proton transport på den tilknyttede EET er deuterium kinetic isotop effekten (KIE). KIE er som endring i elektron overføring kinetics ved utskifting av protoner med deuterium ioner, som representerer virkningen av proton transport elektron overføring kinetics16. Teorien om KIE selv er godt etablert elektrokjemiske målinger med renset enzymer17. Men siden gjeldende produksjon i S. oneidensis MR-1 resultater fra flere forskjellige, og varierende18, kan ikke en bare identifisere EET som hastighetsbegrensning prosessen. For å observere KIE på proton transport prosesser kombinert med lunsj, må vi bekrefte at mikrobielle gjeldende produksjon er begrenset av elektronet transport via OM c- Cyts til elektroden. For dette formålet erstattet vi den supernatant løsningen med frisk medium som inneholder en høy konsentrasjon av laktat som et elektron donor optimal pH og temperaturforhold før KIE måling; Dette erstatning servert to roller: (1) det forbedret frekvensen av oppstrøms metabolske prosesser sammenlignet Fornying, og (2) utelatt svømming cellene i nedbryting fra monolayer biofilm S. oneidensis MR-1 på arbeider elektrode ( Indium tin-dopet oksid (ITO) elektroden). Presenteres detaljert protokollen er ment å hjelpe nye utøvere opprettholde og bekrefte at EET prosessen er rente-bestemme skritt.

<table>
	<tbody>
		<tr>
			<td>Glass cylinder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made, used as the electrochemical reactor</td>
		</tr>
		<tr>
			<td>PTFE cover and base</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made, used as a cover and a foundation of the electrochemical reactor</td>
		</tr>
		<tr>
			<td>Buthyl rubber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made, inserted between each component of electrochemical reactor</td>
		</tr>
		<tr>
			<td>Septa</td>
			<td>GL Science</td>
			<td>3007-16101</td>
			<td>Used as an injection port of electrochemical reactor</td>
		</tr>
		<tr>
			<td>Indium tin-doped oxide (ITO) electrode</td>
			<td>GEOMATEC</td>
			<td>No.0001</td>
			<td>Used as a working electrode, 5&#937;/sq</td>
		</tr>
		<tr>
			<td>Ag/AgCl KCl saturated electrode</td>
			<td>HOKUTO DENKO</td>
			<td>HX-R5</td>
			<td>Used as a reference electrode, &#934;0.30mm</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>The Nilaco Cooporation</td>
			<td>PT-351325</td>
			<td>Used as a counter electrode</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) Broth, Miller</td>
			<td>Becton, Dichkinson and Company</td>
			<td>244620</td>
			<td>Medium for precultivation of S. oneidensis MR-1</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>Becton, Dichkinson and Company</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anthraquinone-1-sulfonate (&#945;-AQS)</td>
			<td>TCI</td>
			<td>A1428</td>
			<td></td>
		</tr>
		<tr>
			<td>Flavin mononucleotide (FMN)</td>
			<td>Wako</td>
			<td>184-00831</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td>Used for defined medium (DM)</td>
		</tr>
		<tr>
			<td>CaCl2 &#183; 2H2O</td>
			<td>Wako</td>
			<td>031-00435</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Wako</td>
			<td>011-03015</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>MgCl2 &#183; 6H2O</td>
			<td>Wako</td>
			<td>135-00165</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)</td>
			<td>DOJINDO</td>
			<td>346-08235</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>Sodium Lactate Solution</td>
			<td>Wako</td>
			<td>195-02305</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>Becton, Dichkinson and Company</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide (D, 99.9%)</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>DLM-4-PK</td>
			<td>Additive for kinetic isotope effect experiments</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>TOKYO RIKAKIKAI CO. LTD.</td>
			<td>LTI-601SD</td>
			<td>Used for precultivation</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>TAITEC</td>
			<td>NR-3</td>
			<td>Used for precultivation</td>
		</tr>
		<tr>
			<td>Autoclave machine</td>
			<td>TOMY SEIKO CO. LTD.</td>
			<td>LSX-500</td>
			<td>Used for sterilization of the electrochemical reactor and the medium</td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>SANYO</td>
			<td>MCV-91BNF</td>
			<td>Used to prevent the contamination of the electrochemical reactor and the medium with other microbes</td>
		</tr>
		<tr>
			<td>Centrifuge separator</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td>Rotational speed upto 6000&#215;g is required</td>
		</tr>
		<tr>
			<td>Nitrogen gas generator</td>
			<td>Puequ CO. LTD.</td>
			<td>PNTN-2</td>
			<td>Nitrogen gas cylinder can also be used instead of gas generator</td>
		</tr>
		<tr>
			<td>UV-vis spectrometer</td>
			<td>SHIMADZU</td>
			<td>UV-1800</td>
			<td>Used for optimization of cell density</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>BioLogic</td>
			<td>VMP3</td>
			<td>Used for biofilm formation and kinetic isotope effect experiments</td>
		</tr>
		<tr>
			<td>Thermal water circulator</td>
			<td>AS ONE</td>
			<td>TR-1A</td>
			<td>Used for maintanance of temperature of electrochemcial reactor</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>HOKUTO DENKO</td>
			<td>HS-201S</td>
			<td>Used for electrochemical experiments</td>
		</tr>
	</tbody>
</table>

electrochemical detection of deuterium kinetic isotope effect on extracellular electron transport in shewanella oneidensis mr-1

Direkte elektrokjemiske påvisning av c-type cytochrome komplekser innebygd i den bakterielle ytre membranen (ytre membran c-skriver cytochrome komplekser; OM c- Cyts) har nylig dukket opp som en roman hele celle analysemetode å karakterisere den bakterielle elektronet transporten fra åndedretts kjeden på cellen utsiden, referert til som den ekstracellulære elektronet transporten (lunsj). Mens den veien og kinetics elektron flyt under Fornying reaksjonen er gransket, har hele celler elektrokjemiske metode å undersøke virkningen av cation transport tilknyttet EET ennå ikke blitt fastsatt. Studien, et eksempel på en biokjemisk teknikk for å undersøke deuterium kinetic isotop effekten (KIE) på EET gjennom OM c- Cyts bruker en modell mikrobe, Shewanella oneidensis MR-1, er beskrevet. KIE på EET prosessen kan oppnås hvis EET gjennom OM c- Cyts fungerer som hastighetsbegrensning trinn i mikrobiell gjeldende produksjon. For dette formål, før en D2O, ble den supernatant løsningen erstattet med friske mediet som inneholder en tilstrekkelig mengde elektron donor støtte frekvensen av oppstrøms metabolske reaksjoner og fjerne planktoniske celler fra en uniform monolayer biofilm på arbeider elektroden. Alternative metoder for å bekrefte hastighetsbegrensningen trinn i mikrobiell gjeldende produksjon som EET gjennom OM c- Cyts er også beskrevet. Våre teknikken av en hel celle elektrokjemiske analysen for å undersøke proton transport kinetics kan brukes på andre electroactive mikrobiell stammer.

Etter 25 h potensielle programmet på 0,4 V (versus hun) dannet en monolayer biofilm på arbeider elektroden ITO glass, tidligere bekreftet av en skanning elektronmikroskop eller en AC confocal mikroskopi4. Representant gang løpet av gjeldende produksjon fra S. oneidensis MR-1 under dannelsen av en monolayer biofilm er vist i figur 2. Selv om gjeldende endrer hver måling, viser produsert gjeldende ikke et avvik over 50% fra ...

Watch this Scientific Journal Video about Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1 at JoVE.com

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Elektrokjemiske gjenkjenning av Deuterium Kinetic isotop effekt på ekstracellulære elektronet Transport Shewanella oneidensis MR-1

Her presenterer vi en protokoll hele celle elektrokjemiske eksperimenter å studere bidrag av proton transport til frekvensen av ekstracellulære elektronet transport via de ytre membran cytochromes i Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Research

JoVE Journal

Biochemistry

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Tilberedning av nukleosomer med Forskjellig Isotop-merket Sister Histoner

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrospray-ionisering Hydrogen-deuterium Veksling massespektrometri for å studere Protein struktur og dynamikk

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

Analysere superkomplekser av den mitokondriale elektrontransportkjede med innfødte elektroforese, in-gel-analyser og elektrolysering

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

En enkel metode for høy gjennomstrømning kjemiske Screening i Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Måle Trans-Plasma membran elektronet Transport av C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Definere Hsp33's Redox regulert Anstandsdame aktivitet og kartlegging Conformational endringer på Hsp33 med Hydrogen-deuterium Exchange massespektrometri

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Prøvetaking og forbehandling av tannemaljen karbonat stabil karbon-og oksygen isotop analyse

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Ekstracellulære Protein Microarray teknologi for høy gjennomstrømning påvisning av lav affinitet reseptor-Ligand interaksjoner

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analyse av β-Amyloid-indusert unormalt Fibrin Clot struktur av spektroskopi og skanning elektronmikroskop

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Analyse av proteinfolding, Transport og nedbrytning i levende celler av radioaktivt puls Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...