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 ...

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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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 ...

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

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