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 arbejde blev økonomisk støttet af en licensbetaling for specielt fremmes forskning fra Japan-samfund til fremme af videnskab (JSP'ER) KAKENHI tilskud antal 24000010, 17H 04969, og JP17J02602, den os Office af Naval Research Global (N62909-17-1-2038). Y.T. er en JSP'ER Research Fellow og støttet af JSP'ER gennem programmet for førende Graduate skoler (MERIT).

1. dannelsen af en éncellelag Biofilm af S. oneidensis hr.-1 på en ITO elektrode (figur 1)
Bemærk: For at forhindre forurening af den elektrokemiske reaktor med andre mikrober, alle medierne, redskaber og dele af den elektrokemiske reaktor skal være steriliseret i forvejen. Når ved hjælp af S. oneidensis hr.-1 celler og konstruere de elektrokemiske reaktorer, alle procedurer bør gennemføres på en ren bænk.
<ol><li>Dyrkning af S. o...

<ol>
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Vores hele-celle elektrokemisk analyse har adskillige tekniske fordele sammenlignet med protein elektrokemi. Mens protein oprensning kræver flere trin tidskrævende procedurer, tager vores hele-celle metode en dag af Self-Organized Biofilmdannelse efter cellekultur. For at opnå en stabil interaktion mellem OM c- Cyts og elektrode, vi behøver kun sterilisation og rengøring af elektrode overflade; Det kræver ikke elektrode ændring for organisering orientering af proteiner4, f.eks....

Elektrokemiske teknikker til direkte karakterisere en redox protein i en intakt bakteriel celle er for nylig opstået siden opdagelsen af metal-reducerende mikrobielle stammer, såsom S. oneidensis hr.-1 eller Geobacter svovlreducens PCA, som har ydre membran c-type cytokrom komplekser (OM c-Cyts) udsat for celle udvendige1,2,3,4,5. OM c- Cyts mægle elektron transport fra den respiratoriske kæde på solid substrater beliggende extracellularly. Denne transport er benævnt ekstracellulære elektron transport (EET)1,6 og er en kritisk proces for nye bioteknologier, såsom mikrobielle brændselsceller6. Derfor, for at forstå de underliggende EET kinetik og mekanismer og dens forbindelse til mikrobiel fysiologi, OM c -Cyts er blevet undersøgt ved hjælp af hele-celle elektrokemi4,7, kombineret med mikroskopi 8 , 9, spektroskopi10,11, og molekylærbiologi2,4. Derimod har at undersøge virkningen af EET-associerede kation transport, fx protoner på EET kinetik i levende celler været næppe fastlaegges metoder, trods proton transport på tværs af bakteriel membraner har en afgørende rolle signalering, homøostase, og energi produktion12,13,14. I nuværende undersøgelse, vi beskriver en teknik til at undersøge virkningen af proton transport på EET kinetik i S. oneidensis hr.-1 celle ved hjælp af hele-celle elektrokemiske målinger, som kræver en identifikation af de hastighedsbegrænsende trin i mikrobielle nuværende produktion15.
En direkte måde at evaluere bidrag af proton transport på den tilknyttede EET er deuterium kinetic isotop effekt (KIE). KIE er observerbare som ændringen i elektron overførsel kinetik ved udskiftning af protoner med deuterium ioner, hvilket svarer til virkningen af proton transport elektron overførsel kinetik16. Teorien om KIE selv er blevet godt etableret, ved hjælp af elektrokemiske målinger med renset enzymer17. Men da nuværende produktion i S. oneidensis hr.-1 resultater fra flere, forskellige og svingende processer18, ikke en blot identificere EET som trafikhastighedsbegrænsning processen. Observere KIE på proton transport processer kombineret med EET, vi har brug at bekræfte, at den mikrobielle nuværende produktion er begrænset af elektron transport via OM c- Cyts til elektrode. Til dette formål erstattet vi den supernatanten løsning med friske medium, der indeholder en høj koncentration af laktat som en elektron donor på optimale pH og temperatur betingelser før KIE måling; denne udskiftning serveret to roller: (1) det forbedret satsen af upstream metaboliske processer i forhold til EET, og (2) udeladt svømning celler i supernatanten frigivet fra éncellelag biofilm af S. oneidensis hr.-1 på arbejde elektrode ( indium tin-doped oxid (ITO) elektrode). Fremlagt detaljerede protokollen er beregnet til at hjælpe nye udøvere fastholde og bekræfte, at EET proces er det hastighedsbestemmende trin.

<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 elektrokemisk detektion af c-Skriv cytokrom komplekser indlejret i den bakterielle ydre membran (ydre membran c-Skriv cytokrom komplekser; OM c- Cyts) har for nylig dukket op som en roman hele-celle analytisk metode til at karakterisere den bakterielle elektron transport fra respiratorisk kæden til celle udvendige, kaldes den ekstracellulære elektron transport (EET). Mens pathway og kinetik af elektron flow under EET reaktion er blevet undersøgt, er en helhed-celle elektrokemiske metode til at undersøge virkningen af kation transport forbundet med EET endnu ikke fastlagt. I den foreliggende undersøgelse, et eksempel på en biokemisk teknik til at undersøge deuterium kinetic isotop effekt (KIE) på EET gennem OM c- Cyts ved hjælp af en model mikrobe, Shewanella oneidensis hr.-1, er beskrevet. KIE på EET processen kan opnås, hvis EET gennem OM c- Cyts fungerer som den trafikhastighedsbegrænsning trin i den mikrobielle nuværende produktion. Herpå, før tilsætning af D2O, blev supernatanten løsningen erstattet med friske medier indeholdende en tilstrækkelig mængde af elektron donoren støtte sats af upstream metaboliske reaktioner og fjerne de planktoniske celler fra en ensartet éncellelag biofilm på arbejde elektrode. Alternative metoder til at bekræfte hastighedsbegrænsende trin i mikrobiel nuværende produktion som EET gennem OM c- Cyts er også beskrevet. Vores teknik af en helhed-celle elektrokemisk analyse for at undersøge proton transport kinetik kan anvendes til andre electroactive mikrobielle stammer.

Efter 25 h af potentielle anvendelse på +0.4 V (versus hun), blev en éncellelag biofilm dannet på den arbejdende elektrode af ITO glas, som var tidligere bekræftet af enten en scanning elektronmikroskopi eller en Konfokal mikroskopi4. De repræsentative tidsforløb af nuværende produktion fra S. oneidensis hr.-1 under dannelsen af en éncellelag biofilm er vist i figur 2. Selv om nuværende ændrer i hver måling, udviser...

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

Elektrokemisk detektion af Deuterium Kinetic isotop effekt på ekstracellulære elektron Transport i Shewanella oneidensis hr.-1

Her præsenterer vi en protokol af hele-celle elektrokemiske eksperimenter til at studere bidrag af proton transport til sats af ekstracellulære elektron transport via de ydre membran cytokromer, der i Shewanella oneidensis hr.-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

Rekonstituering af nucleosomer med differentielt isotopmærkede 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 elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

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

Analyse af superkomplekser af den mitokondrieelektron transportkæde med indfødte 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 simpel metode til High Throughput kemisk 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

Måling af Trans-Plasma membran elektron Transport af 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-regulerede anstandsdame aktivitet og kortlægning konformationelle ændringer på Hsp33 ved hjælp af brint-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

Prøveudtagning og forbehandling af tand emalje carbonat for stabil Carbon og Oxygen isotop analyser

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 til High Throughput påvisning af lav affinitet Receptor-Ligand interaktioner

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 af β-Amyloid-induceret afvigelser Fibrin blodprop strukturen af spektroskopi og Scanning elektronmikroskopi

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 af proteinfoldning, Transport og nedbrydning i levende celler af radioaktivt puls Chase

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