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

Dit werk werd financieel ondersteund door een Grant-in-Aid voor speciaal bevorderd onderzoek van de vereniging van Japan voor promotie van wetenschap (JSPS) KAKENHI Grant nummer 24000010, 17H 04969, en JP17J02602, de ons kantoor van Naval Research Global (N62909-17-1-2038). Y.T. is een JSPS Research Fellow en ondersteund door JSPS via het programma voor toonaangevende gediplomeerde scholen (MERIT).

1. de vorming van een Biofilm Monolayer van S. oneidensis heer-1 op een ITO elektrode (Figuur 1)
Opmerking: Om te voorkomen dat de besmetting van de elektrochemische reactor met andere microben, alle media, werktuigen, en onderdelen van de elektrochemische reactor moeten worden gesteriliseerd bij voorbaat. Wanneer cuvetten van S. oneidensis heer-1 en de bouw van de elektrochemische reactoren, alle procedures moeten worden uitgevoerd op een schone b...

<ol>
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J., 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=Redox+Linked+Flavin+Sites+in+Extracellular+Decaheme+Proteins+Involved+in+Microbe-Mineral+Electron+Transfer.">Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.</a> Sci. Rep. 5, 11677 (2015).</li><li>Saito, J., Hashimoto, K., Okamoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavin+as+an+Indicator+of+the+Rate-Limiting+Factor+for+Microbial+Current+Production+in+Shewanella+oneidensis+MR-1.">Flavin as an Indicator of the Rate-Limiting Factor for Microbial Current Production in Shewanella oneidensis MR-1.</a> Electrochim. Acta. 216, 261-265 (2016).</li><li>Guo, J. 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Onze geheel-cel elektrochemische bepaling heeft een aantal technische voordelen vergeleken met eiwit elektrochemie. Hoewel eiwitreiniging vereist de multi-step tijdrovende procedures, neemt onze geheel-cell, methode een dag van zelf-georganiseerde biofilm vorming na celkweek. Om te bereiken van een stabiele interactie tussen OM c- Cyts en de elektrode, moeten we alleen sterilisatie en reiniging van het oppervlak van de elektrode; het vereist geen wijziging van de elektrode voor het organiseren van de richting va...

Elektrochemische technieken te karakteriseren direct een redox-eiwit in een intact bacteriële cel onlangs naar voren zijn gekomen sinds de ontdekking van metaal-reducerende microbiële stammen, zoals S. oneidensis heer-1 of Geobacter sulfurreducens partnerschaps-en samenwerkingsovereenkomst, die hebben de buitenmembraan cytochroom c-type complexen (OM c-Cyts) blootgesteld aan de cel exterieur1,2,3,4,5. De OM- c- Cyts bemiddelen elektronentransport uit de luchtwegen keten aan vaste substraten gelegen extracellularly. Dit vervoer wordt hierna aangeduid als extracellulaire elektronentransport (EET)1,6 en is een kritiek proces voor opkomende biotechnologie, zoals microbiële brandstofcellen6. Daarom, om te begrijpen van de onderliggende EET-kinetiek en mechanismen, en haar link naar microbiële fysiologie, OM c -Cyts zijn onderzocht met behulp van geheel-cel elektrochemie4,7, gecombineerd met microscopie 8 , 9, spectroscopie10,11, en moleculaire biologie2,4. In tegenstelling, zijn methoden voor het onderzoeken van de effecten van het EET-geassocieerde catie vervoer, bijvoorbeeld protonen, op EET kinetiek in levende cellen nauwelijks vastgesteld, ondanks proton vervoer over bacteriële membranen hebben een cruciale rol signalering, homeostase en energie productie12,13,14. In de huidige studie, beschrijven we een techniek om de onderzoeken van het effect van het proton vervoer op EET kinetiek in de S. oneidensis heer-1 cel met behulp van geheel-cel Electrochemische metingen, waarvoor de identificatie van de snelheidslimieten stap in microbiële huidige productie15.
Een directe manier om de bijdrage van het proton vervoer op de bijbehorende EET is het deuterium kinetisch-isotoopeffect (KIE). De KIE is waarneembaar als de verandering in de elektronen overdracht kinetiek op de vervanging van protonen met deuterium ionen, die de impact van het proton vervoer op elektron overdracht kinetiek16aangeeft. De theorie van KIE zelf is goed opgezet Electrochemische metingen met gezuiverde enzymen17. Echter, aangezien huidige productie in S. oneidensis heer-1 wordt veroorzaakt door meerdere, divers, en schommelende processen18, niet één EET gewoon identificeren als de snelheidslimieten proces. Om te zien hoe de KIE op proton transportprocessen EET wordt gekoppeld, moeten we om te bevestigen dat de microbiële huidige productie wordt beperkt door elektronentransport via OM c- Cyts op de elektrode. Voor dit doel vervangen wij het supernatant oplossing met verse opslagmedium met een hoge concentratie van lactaat als een elektrondonor tegen de optimale pH en temperatuurvoorwaarden voor KIE meting; deze vervanging diende twee rollen: (1) het verbeterd het tarief van de upstream metabole processen ten opzichte van de EET, en (2) de cellen van het zwemmen in het supernatant vrijgelaten uit de biofilm enkelgelaagde van S. oneidensis heer-1 op de werkende elektrode (weggelaten indium tin-doped oxide (ITO) elektrode). De gepresenteerde gedetailleerd protocol is bedoeld om te helpen nieuwe beoefenaars handhaven en bevestigen dat het proces EET de tarief-bepalen stap is.

<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

Directe elektrochemische detectie van c-type cytochroom complexen ingebed in de bacteriële buitenmembraan (buitenmembraan c-Typ cytochroom complexen; OM c- Cyts) heeft onlangs naar voren gekomen als een roman geheel-cel analysemethode te karakteriseren de bacteriële elektronentransport uit de luchtwegen keten aan de buitenkant van de cel, hierna aangeduid als de extracellulaire elektronentransport (EET). Terwijl het traject en de kinetiek van de stroom van elektronen tijdens de EET-reactie zijn onderzocht, is een elektrochemische geheel-cell, methode te onderzoeken van de effecten van het vervoer van de catie gekoppeld EET nog niet vastgesteld. In de huidige studie, een voorbeeld van een biochemische techniek te onderzoeken het deuterium kinetisch-isotoopeffect (KIE) op EET t/m OM c- Cyts met behulp van een model-microbe, Shewanella oneidensis heer-1, wordt beschreven. De KIE op het EET-proces kan worden verkregen als de EET t/m OM c- Cyts als de snelheidslimieten stap in de microbiële huidige productie fungeert. Te dien einde, vóór de toevoeging van D2O, werd de bovenstaande oplossing vervangen door verse medium met een voldoende hoeveelheid van de elektronendonor ter ondersteuning van het tempo van de upstream metabole reacties, en te verwijderen van de planktonische cellen van een uniform enkelgelaagde biofilm op de elektrode werken. Alternatieve methoden te bevestigen de snelheidslimieten-stap in microbiële huidige productie als EET t/m OM c- Cyts ook worden beschreven. Onze techniek van een geheel-cel elektrochemische assay voor het onderzoeken van proton vervoer kinetiek kan worden toegepast op andere microbiële stammen van electroactive.

Na 25 h van potentiële toepassing op +0.4 V (in tegenstelling tot ze) ontstond een enkelgelaagde biofilm op de elektrode van de werken van ITO glas, die eerder door een scanning elektronen microscopie of een confocale microscopie4werd bevestigd. De representatieve tijdsverloop van de huidige productie van de S. oneidensis heer-1 tijdens de vorming van een biofilm enkelgelaagde is afgebeeld in Figuur 2. Hoewel de huidige in el...

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

Elektrochemische detectie van Deuterium kinetisch-isotoopeffect op extracellulaire elektronentransport in Shewanella oneidensis heer-1

Hier presenteren we een protocol van geheel-cel elektrochemische experimenten te bestuderen van de bijdrage van proton vervoer aan het tarief van extracellulaire elektronentransport via de buitenmembraan cytochromes complex in Shewanella oneidensis heer-1.

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

The overall goal of this protocol is to observe the contribution of proton transport to the rate of bacterial extracellular electron transport by the detection of the deuterium kinetic isotope effect. Our method reveals the impact of proton transport on the kinetics of extracellular electron transport or EET from bacteria to an electrode via outer membrane c-type cytochromes. Our assay reflects the native proton management in EET because we used living bacteria rather than purified enzyme.However, our challenge was to remove the noises from other cellular processes. The deuterium kinetic isotope effect termed as KIE is one of the best technique to examine the contribution of proton transport on the associated electron transport process. To grow Shewanella MR-1 cells, transfer one colony of the cells grown on an agar plate into 15 milliliters of LB medium.Grow the cells at 30 degrees Celsius for 24 hours in aerobic conditions with shaking at 160 rpm. Centrifuge the cell suspension at 6, 000 times gravity for 10 minutes. Then, resuspend the resultant cell pellet in 15 milliliters of Defined Medium, supplemented with 10 millimolar lactate as the source of carbon and 0.5 grams per liter yeast extract as trace elements for the cells.After further cultivating the cell suspension aerobically at 30 degrees Celsius for 12 hours with shaking at 160 rpm, centrifuge again at 6, 000 times gravity for 10 minutes. Wash the resultant cell pellet twice with DM medium by centrifugation for 10 minutes at 6, 000 times gravity before the electrochemical experiment. To construct a three-electrode electrochemical reactor, put an ITO substrate as the working electrode at the bottom of the reactor.Subsequently, insert a glass cylinder and a polytetrafluoroethylene cover and insert a platinum wire as the counter-electrode. Then, insert a silver chloride electrode into the reactor as the reference electrode. Next, add four milliliters of DM supplemented with 10 millimolar lactate and 0.5 grams per liter yeast extract into the electrochemical reactor.After confirming that there is no leakage from the electrochemical reactor, flow the nitrogen gas into the electrochemical reactor over 20 minutes to maintain anerobic conditions inside the electrochemical reactor. Connect the electrochemical reactor to a potentiostat and apply a positive 0.4 volts to the ITO electrode, keeping the temperature of the electrochemical reactor at 30 degrees Celsius using an external water circulation system. To perform electrochemical cultivation, first adjust the cell density of the suspension to an optical density of 1.43 at 600 nanometers with DM supplemented by 10 millimolar lactate and 0.5 grams per liter yeast extract.Add 0.3 milliliters of the cell suspension into the electrochemical reactor through the injection port using a syringe. The OD 600 in the reactor changes to 0.1. Continue the potential application at positive 0.4 volts to the ITO electrode for 25 hours.Stop the potential application and disconnect the electrochemical reactor from the potentiostat and the water circulation system. Flow the nitrogen gas into a bottle containing DM with 10 millimolar lactate over 20 minutes to remove oxygen from the medium. Now, extremely slowly and gently, remove all the supernatant from inside the electrochemical reactor using a syringe under flowing nitrogen gas.Then, add four milliliters of fresh DM containing 10 millimolar lactate using a syringe. Slant the electrochemical reactor to remove all of the supernatant on the wall of the reactor. Repeat these steps three times in total.Stop the gas flow and connect the electrochemical reactor to the potentiostat again, applying a positive 0.4 volts to the ITO electrode at 303 Kelvin. Confirm that the current production from a monolayer biofilm of Shewanella MR-1 is stable and does not increase rapidly. If the current increases steeply, wait until the current stabilizes with a 5%increase over 10 minutes.Extremely slowly and gently, add 40 microliters of anoxic 50 volume percent D2O into the electrochemical reactor using a syringe such that the concentration is 0.5 volume percent D2O in the reactor. To prevent damage to the biofilm by D20 addition, inject the D2O dropwise. Wait for the current to stabilize and subsequently add the D2O up to 4.4 volume percent to obtain the KIE value, which is the ratio of current production and presence of D2O and H2O.Check the effect of the same volume of H2O addition on the current production. Here, the solid line is the representative result for the microbial current change induced by D2O addition. Addition of D2O sharply decreased the microbial current within 10 seconds, while H20 had almost no current decrease as shown with the dotted line.To confirm that the observed current decrease by D2O is attributable to electron transport through outer membrane cytochromes, a diffusing electron mediator was used. Here addition of 100 micromolar alpha-AQS enhanced the current production by more than five times and the current production remained unaffected by D2O addition. These results indicate that the kinetics of the metabolic reactions are fast enough so that the extracellular electron transfer process is rate-limiting.An alternative method for assessing this is the addition of the flavin molecule into the electrochemical reactor. An instant anodic current increase by flavin addition indicates rate-limitation by the electron transport process via outer membrane cytochromes. After watching this video, you should have a good understanding of how to examine the kinetic isotope effect on the extracellular electron transport process.The most critical point to successfully obtain the kinetic isotope effect is to clarify and confirm the condition where the electron transport through the outer membrane cytochromes limits the rate of current production. In this video, we introduced two methods to confirm this condition, observation of current change by addition of flavin and by addition of diffusing extra mediator, such as alpha-AQS. Once the rate-limitation is confirmed, this system can be used not only for the deuterium experiment but also for the biochemical characterization of outer membrane cytochromes.Furthermore, this protocol could be applicable to examining other electroactive microbes, as long the extracellular electron transport process is rate-limiting. Our methodology provides a basic and general technique to investigate the extracellular electron transport process via outer membrane cytochromes in vivo.

Research

JoVE Journal

Biochemistry

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

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Meten van Trans-Plasma membraan elektronentransport door C2C12 Myotubes

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