이 작품 과학 진흥 (JSP) KAKENHI 보조금 번호 24000010, 17 H 04969, 일본 사회에서 특별히 추진 연구 및 JP17J02602, 미국 사무실의 해군 연구 글로벌 (N62909-17-1-2038)는 선진적인 재정적으로 지원 했다. Y.T.는 JSP 연구원 이며 프로그램을 통해 JSP에 대 한 선도 대학원 학교 (공로) 지원.

1. ITO 전극 (그림 1)에 S. oneidensis 씨-1의 단층 Biofilm의 형성
참고: 다른 미생물과 전기 화학 반응 기의 오염을 방지 하기 위해, 모든 미디어, 구현 및 전기 화학 반응 기의 구성 한다 살 균 되어야 사전에. 때 깨끗 한 벤치에 모든 절차를 지휘 한다 S. oneidensis 씨-1 셀을 사용 하 고 전기 원자로 건설.
<ol><li>S. oneidensis 씨-1 셀의 재배 참고: S. oneidensis 씨-1의 단층 biofilm 형성 되었다는 이토에는 조건에 따라 전극 보고 이전4.<ol>
<li>S. oneidensis 씨-1 세포 성장, S. oneidensis 씨-1 파운드 매체 (20 g/L) 160 rpm에서 떨고 호 기성 조건에서 24 시간에 30 ° C에서의 15 mL에 Luria Bertani (파운드) (20 g/L) 및 bacto agar (15 g/L)로 구성 된 한 천 배지에서 성장 한 식민지를 추가 합니다.</li>
		<li>10 분 동안 6000 × g 에서 세포 현 탁 액을 원심 및 정의 된 매체 (pH 7.8)의 15 mL에 결과 셀 펠 릿 resuspend (DM: NaHCO3 [2.5 g/L], CaCl2·2H2O [0.08 g/L], NH4Cl [1.0 g/L] MgCl2· 6 H2O [0.2 g/L], NaCl [10 g/L], 그리고 2-[4-(2-hydroxyethyl)-1-piperazinyl] [HEPES; 7.2 g/L] ethanesulfonic 산), 젖 산 10 m m와 0.5 g/L 효 모 추출 물, 탄소 소스로 보충 및 S. oneidensis 씨-1에 대 한 추적 요소 각각.</li>
		<li>또한 160 rpm에서 떨고와 12 h 30 ° C에서 aerobically 세포 현 탁 액을 배양 하 고 원심 다시 10 분 세척을 위한 6000 × g 에서 DM 매체와 두 번 결과 셀 펠 릿은 화학 전에 6000 × g에서 10 분 원심 분리 하 여 실험입니다.</li>
	</ol>
</li>
	<li>3-전극 전기 화학 반응 (그림 1)의 건설<ol>
<li>반응 기의 하단에 작업 전극으로 ITO 기판을 넣어.</li>
		<li>그 후, 삽입 유리 실린더 (2 cm의 직경) 및 소계 (PTFE) 커버. 그런 다음 삽입 Ag/AgCl (포화 KCl)와 백 금 철사는 원자로에 참조 및 카운터 전극으로 각각. 참고: 공기 및 솔루션의 누설을 방지 하기 위해 각 구성 요소 간의 부 틸 고무 시트를 삽입 합니다.</li>
		<li>DM의 4.0 mL 10mm 젖 산으로 보충 하 고 0.5 g/L 효 모 추출 화학 반응 기에 추가 합니다.</li>
		<li>전기 화학 반응 기에서 누설 확인, 전기 화학 반응 화학 반응 기 내부의 혐 기성 조건을 유지 하기 위해 20 분 이상으로 질소 가스 흐름. 참고: 다른 미생물 오염을 방지 하기 위해, 가스 한다 필터링 전에 그것은 전기 화학 반응으로 흐른다.</li>
		<li>전기 화학 반응 기는 potentiostat에 연결 하 고 적용 0.4 V (표준 수소 전극 대 그녀) ITO 전극에는 외부 물 순환 시스템을 사용 하 여 30 ° C에서 전기 화학 반응 기의 온도 유지. 참고: 외부 전기 분야의 영향을 방지 하기 위해, 전기 화학 반응 기는 패러데이 케이지에 배치 되어야 합니다.</li>
	</ol>
</li>
	<li>S. oneidensis 씨-1 세포 (그림 1 및 그림 2)의 전기 화학 경작<ol>
<li>1.1 600 1.43의 광학 밀도에 단계에서 얻은 서 스 펜 션의 셀 밀도 조정 10 m m 0.5 g/L 및 젖 산 효 모에 의해 보충 하는 dm nm (OD600) 추출. 참고: 올바른 세600를, 1.43 OD600 조정 전에 대 한 UV 분석기에 OD600 ≤ 0.8의 세포 현 탁 액을 적용 됩니다.</li>
		<li>세포 현 탁 액의 0.3 mL 주사기를 사용 하 여 주입 포트를 통해 전기 화학 반응 기에 추가: 원자로에서 OD600 0.1로 변경. 참고: OD600 와 0.3 mL 셀 서 스 펜 션의 추가 = 1.43 OD600 와 솔루션의 4.3 ml에서 4.0 mL 중간 결과 포함 하는 전기 원자로에 0.1 =. 다른 원자로 사용 하 여 다른 볼륨, 셀 밀도의 계산이 필요 합니다.</li>
		<li>0.4 V (그녀) 대 25 h ITO 전극에 잠재적인 응용 프로그램을 계속 합니다. 참고: ITO 전극에 단층 biofilm의 형성에 대 한 생산된 전류 전시 그림 2에서 50% 미만의 편차 확인 합니다.</li>
	</ol>
</li>
</ol>2. 젖 산 10 m m (그림 3)와 신선한 DM 매체와 상쾌한의 교체
<ol><li>잠재적인 응용 프로그램을 중지 하 고 전기 화학 반응 기는 potentiostat와 물 순환 시스템에서 분리 합니다.</li>
	<li>10 m m와 신선한 DM 매체와 상쾌한의 대체 젖이 나올<ol>
<li>DM를 포함 하는 매체에서 산소를 제거 하 10mm 젖 산 20 분 이상으로 병에 질소 가스 흐름.</li>
		<li>천천히 흐르는에서 주사기를 사용 하 여 전기 화학 반응 기 내부의 모든 상쾌한 제거 질소 가스 (그림 3a, b). 참고: 질소 가스에 의해 ITO 전극에는 biofilm를 위반을 피하기 위해, 가스 액체 표면 위의 흐름 해야 합니다.</li>
		<li>(그림 3c) 주사기를 사용 하 여 10mm 젖 산을 포함 하는 신선한 DM의 4.0 mL를 추가 합니다. 참고: ITO 전극에는 biofilm를 위반을 피하기 위해, 전기 화학 반응 기의 벽을 따라 중간을 주사 천천히. 유지 하기 위해 젖은 biofilm, 매체 상쾌한의 제거 후 즉시 추가 되어야 합니다. 상쾌한의 제거 후 중간까지 1 분의 주입 S. oneidensis 씨-1는 biofilm에서 현재 생산을 영향을 주지 않습니다.</li>
		<li>전기 화학 반응 기의 벽에 부착 된 상쾌한의 모두 제거를 전기 화학 반응 기 경사</li>
		<li>총에 세 번 단계 2.2.2-2.2.4를 반복 합니다.</li>
	</ol>
</li>
	<li>가스 흐름을 중지 하 고 potentiostat 다시, 303 공화국에서 ITO 전극 (그녀) 대 0.4 V 적용에 전기 화학 반응 기를 연결</li>
</ol>3. 동유럽 표준시 과정 (그림 4)에 인기를 측정 하는 중수소 물 추가
<ol><li>S. oneidensis 씨-1의 단층 biofilm에서 현재 생산 안정적 이며 빠른 속도로 증가 하지 않습니다 확인 합니다. 현재 가파르게 증가 하는 경우 현재 5% 증가 10 분 이상에서 안정화 될 때까지 기다립니다. 참고: 다른 미생물 긴장의 오염 없이 ITO 전극에 단층 biofilm의 형성 확인 되었다 rDNA 시퀀스와 ITO 전극에 biofilm의 스캐닝 전자 현미경 이미지를 보고 이전4. 동유럽 표준시 프로세스 속도 제한의 확인에 대 한 추가 왕복 전자 중재자 100 µ M 안트라퀴논-1-된 α-AQS () 등의 효과 모니터링 합니다. 15 대 한 자세한 내용은 참조 및 대표적인 결과 섹션을 참조 하십시오.</li>
	<li>농도 0.5% (v/v) D2O 원자로에 주사기를 사용 하 여 전기 화학 반응으로 무산 소 50% (v/v) D2O의 40 µ L를 추가 합니다. 참고: D2O 추가 의해는 biofilm 손상을 방지, 주사 D2O drop-wise.</li>
	<li>전류 안정화를 기다립니다 하 고 이후 추가 D2O 4.0% (v/v). 참고: KIE 값 (D2O 및 H2O의 현재 생산의 비율)를, 현재 생산에 H2O 추가의 동일한 볼륨의 효과 확인 하십시오.</li>
</ol>

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B., 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=Reduction+of+Cr(VI)+by+Escherichia+coli+BL21+in+the+presence+of+redox+mediators.">Reduction of Cr(VI) by Escherichia coli BL21 in the presence of redox mediators.</a> Bioresource Technol. 123, 713-716 (2012).</li><li>Nealson, K., Saffarini, D., Moser, D., Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Spectrophotometric+Method+for+Monitoring+Tactic+Responses+of+Bacteria+under+Anaerobic+Conditions.">A Spectrophotometric Method for Monitoring Tactic Responses of Bacteria under Anaerobic Conditions.</a> J Microbiol. Meth. 20 (3), 211-218 (1994).</li><li>Myers, C. R., Myers, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+exposure+of+the+outer+membrane+cytochromes+of+Shewanella+oneidensis+MR-1.">Cell surface exposure of the outer membrane cytochromes of Shewanella oneidensis MR-1.</a> Lett. Appl. Microbiol. 37 (3), 254-258 (2003).</li><li>Lower, B. H., 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=Antibody+Recognition+Force+Microscopy+Shows+that+Outer+Membrane+Cytochromes+OmcA+and+MtrC+Are+Expressed+on+the+Exterior+Surface+of+Shewanella+oneidensis+MR-1.">Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1.</a> Appl. Environ. Microbiol. 75 (9), 2931-2935 (2009).</li><li>Chen, X. X., Ferrigno, R., Yang, J., Whitesides, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+properties+of+cytochrome+c+adsorbed+on+self-assembled+monolayers:+A+probe+for+protein+conformation+and+orientation.">Redox properties of cytochrome c adsorbed on self-assembled monolayers: A probe for protein conformation and orientation.</a> Langmuir. 18 (18), 7009-7015 (2002).</li><li>McMillan, D. G. G., 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+impact+of+enzyme+orientation+and+electrode+topology+on+the+catalytic+activity+of+adsorbed+redox+enzymes.">The impact of enzyme orientation and electrode topology on the catalytic activity of adsorbed redox enzymes.</a> Electrochim. Acta. 110, 79-85 (2013).</li><li>Dinh, H. T., 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=Iron+corrosion+by+novel+anaerobic+microorganisms.">Iron corrosion by novel anaerobic microorganisms.</a> Nature. 427 (6977), 829-832 (2004).</li><li>McGlynn, S. E., Chadwick, G. L., Kempes, C. P., Orphan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+activity+reveals+direct+electron+transfer+in+methanotrophic+consortia.">Single cell activity reveals direct electron transfer in methanotrophic consortia.</a> Nature. 526 (7574), 531-535 (2015).</li><li>Okamoto, A., Nakamura, R., Nealson, K. H., Hashimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bound+Flavin+Model+Suggests+Similar+Electron-Transfer+Mechanisms+in+Shewanella+and+Geobacter.">Bound Flavin Model Suggests Similar Electron-Transfer Mechanisms in Shewanella and Geobacter.</a> Chemelectrochem. 1 (11), 1808-1812 (2014).</li><li>Okamoto, A., Hashimoto, K., Nealson, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavin+Redox+Bifurcation+as+a+Mechanism+for+Controlling+the+Direction+of+Electron+Flow+during+Extracellular+Electron+Transfer.">Flavin Redox Bifurcation as a Mechanism for Controlling the Direction of Electron Flow during Extracellular Electron Transfer.</a> Angew. Chem. Int. Ed. 53 (41), 10988-10991 (2014).</li><li>Tokunou, Y., 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=Acceleration+of+Extracellular+Electron+Transfer+by+Alternative+Redox-Active+Molecules+to+Riboflavin+for+Outer-Membrane+Cytochrome+c+of+Shewanella+oneidensis+MR-1.">Acceleration of Extracellular Electron Transfer by Alternative Redox-Active Molecules to Riboflavin for Outer-Membrane Cytochrome c of Shewanella oneidensis MR-1.</a> J Phys. Chem. C. 120 (29), 16168-16173 (2016).</li><li>Rowe, A. R., 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=Tracking+electron+uptake+from+a+cathode+into+Shewanella+cells:+implications+for+generating+maintenance+energy+from+solid+substrates.">Tracking electron uptake from a cathode into Shewanella cells: implications for generating maintenance energy from solid substrates.</a> bioRxiv. , 116475 (2017).</li></ol>

저자는 공개 없다.

우리의 전체 셀 전기 화학 분석 결과 단백질 전기에 비해 몇 가지 기술적인 이점이 있다. 단백질 정화 다단계 시간이 걸리는 절차 필요, 우리의 전체 셀 메서드 자체 조직된 biofilm 형성 세포 배양 후의 어느 날 걸립니다. 톰 c간의 안정적인 상호 작용을 달성 하기 위해-대련 청 려 고 전극, 우리 필요만 살 균과 청소의 전극 표면; 톰 c를 향하는 동안, 전극에 부착 단백질4</su...

직접 하는 그대로 세균 세포에서 산화 환 원 단백질 전기 기술을 최근 금속 감소 미생물 긴장, S. oneidensis 씨-1 등 Geobacter sulfurreducens PCA의 발견 이후 등장 외부 막 시 토 크롬 c 형 단지 (OM c-대련 청 려) 셀 외부1,2,3,,45에 노출 되어 있다. 톰 c-대련 청 려 호흡기 체인에서 extracellularly 있는 고체 기판에 전자 수송 중재. 이 전송 extracellular 전자 전송 (동유럽 표준시)1,6 이라고 하 고 미생물 연료 전지6등 신흥 바이오에 대 한 중요 한 과정 이다. 따라서, 기본 동유럽 표준시 속도 론 및 메커니즘 및 미생물 생리학에 그것의 연결을 이해 하 옴 c-대련 청 려 되었습니다 조사 전체 셀 전기 화학4,7, 현미경과 결합을 사용 하 여 8 , 9, 분광학10,11, 그리고 분자 생물학2,4. 반면, 동유럽 표준시 관련 된 양이온 수송, 예를 들어, 양성자, 살아있는 세포에서 동유럽 표준시 활동에 미치는 영향을 조사 하는 방법 부족 하 게 설립 되어, 세균 막 중요 한 역할을가지고 걸쳐 양성자 수송에도 불구 하 고 신호, 항상성, 및 에너지 생산12,,1314. 현재의에서 연구, 우리는 양성자 수송 필요의 속도 제한 단계 식별 전체 셀 전기 화학 측정을 사용 하 여 S. oneidensis 씨-1 셀에 동유럽 표준시 활동에의 영향을 검사 하는 기법을 설명 미생물 현재 생산15.
1 직접 연결 된 동유럽 표준시에 양성자 수송의 기여를 평가 방법은 중수소 활동적인 동위 원소 효과 (KIE)입니다. 인기의 전자 전송 속도 론16양성자 교통 영향을 나타내는 중수소 이온, 양성자의 교체 시 전자 전송 속도에서 변화 관찰입니다. KIE 자체의 이론은 잘 설립 되었습니다 정제 효소17전기 화학 측정을 사용 하 여. 그러나, S. oneidensis 씨-1에 현재 생산, 여러 다양 한, 그리고 변동 과정18에서 결과, 이후 하나 확인할 수 없습니다 단순히 동유럽 표준시 속도 제한 과정으로. 관찰 하기 위해 동유럽 표준시 함께 양성자 전송 프로세스에 인기, 우리는 미생물 현재 생산 옴 c통해 전자 전송에 의해 제한 됩니다 확인 해야-대련 청 려 전극에. 이 목적을 위해 우리 신선한 매체에서 최적 pH 및 온도 조건 KIE 측정; 전에 전자 기증자로 젖 산의 높은 농도 포함 하는 표면에 뜨는 솔루션 대체 이 교체 봉사 두 역할: (1) 동유럽 표준시에 비해 업스트림 신진 대사 과정의 속도 향상 하 고 (2) 작업 전극 (에 S. oneidensis 씨-1의 단층 biofilm에서 발표 하는 상쾌한 수영 셀 생략 인듐 주석 첨가 산화물 (ITO) 전극). 새로운 실무자 유지 하 고 동유럽 표준시 프로세스 속도 결정 단계는 확인을 있도록 제시 상세한 프로토콜 것입니다.

<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

직접 c의 전기 화학 검출-시 토 크롬 단지 세균 외부 막에 포함 된 입력 (외부 막 c-입력 시 토 크롬 복합물; 톰 c-대련 청 려)는 최근 소설 전체 셀 셀 외관 호흡기 체인에서 세균성 전자 수송 특성 분석 방법으로, 세포 외 전자 전송 (동유럽 표준시)로. 통로 동유럽 표준시 반응 동안 전자 흐름의 속도, 조사 하는 동안 전체 셀 전기 메서드 동유럽 표준시와 관련 된 양이온 수송의 영향 검토를 하지 아직 설립 되었습니다. 현재 연구에서 중수소 활동적인 동위 원소 효과 (KIE) 동유럽 표준시에 옴 c를 검사 하는 생 화 확 적인 기술의 예-대련 청 려 모델 미생물, Shewanella oneidensis 미스터-1을 사용 하 여 설명 되어 있습니다. 동유럽 표준시 프로세스에 KIE 옴 c통해 동유럽 표준시-대련 청 려 미생물 현재 생산에서 속도 제한 단계 역할을 하는 경우에 얻어질 수 있다. 끝으로, D2O의 추가 하기 전에 표면에 뜨는 솔루션 업스트림 신진 대사 반응의 속도 지원 하 고 유니폼에서 planktonic 세포를 제거 하는 전자 기증자의 충분 한 금액을 포함 하는 신선한 미디어로 대체 되었습니다. 작업 전극에 단층 biofilm입니다. 속도 제한 확인 대체 방법을 미생물 현재 생산에서 단계 옴 c통해 동유럽 표준시-대련 청 려도 설명 됩니다. 양성자 전송 속도 론 조사를 위한 전기 화학 분석 결과 전체 셀의 우리의 기술은 다른 electroactive 미생물 계통에 적용할 수 있습니다.

0.4 V (그녀) 대에서 잠재적인 응용 프로그램의 25 h, 후 단층 biofilm 이전 스캐닝 전자 현미경 검사 법 또는4confocal 현미경 검사 법 의해 확인 되었다 이토 유리의 작업 전극에 형성 되었다. 단층 biofilm의 형성 중 S. oneidensis 씨-1에서 현재 생산의 대표적인 시간 과정은 그림 2에 표시 됩니다. 비록 현재는 모든 측정에 변경, 단층 biofi...

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

전기 화학 탐지의 중수소 활동적인 동위 원소 효과 Shewanella oneidensis 미스터-1에서 세포 외 전자 전송에

여기 선물이 전체 셀 전기 화학 실험 Shewanella oneidensis 미스터-1에서 복잡 한 외부 막 한다 통해 extracellular 전자 전송의 속도를 양성자 수송의 기여를 공부 하는 프로토콜.

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

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Biochemistry

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

9.9K 조회수.  University of Tokyo. 여기 선물이 전체 셀 전기 화학 실험 Shewanella oneidensis 미스터-1에서 복잡 한 외부 막 한다 통해 extracellular 전자 전송의 속도를 양성자 수송의 기여를 공부 하는 프로토콜.

비디오: 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

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

된 유의 동위 원소 표지 자매 히스톤과 뉴 클레오의 재구성

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

연구

생화학

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

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

시간 해결 전기 분무 이온화 수소 - 중수소 교환 질량 분석 단백질 구조와 역학 연구를위한

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

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

네이티브 전기 영동, In-gel Assay 및 Electroelution으로 미토콘드리아 전자 수송 사슬의 수퍼 콤플렉스 분석

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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

펩 티 드 선호도 시 약 검색 및 분석 결과 분리의 세균성 디스플레이 라이브러리의 반자동된 Biopanning

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

꼬마 선 충 에서 높은 처리량 화학 심사 하는 간단한 방법

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

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 &#197;, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.

Negative stain EM is a powerful technique for visualizing macromolecular structure, but different staining techniques can produce varying results in a sample dependent manner. Here several negative staining approaches are described in detail to provide an initial workflow for tackling the visualization of challenging systems.

유사에 부정적인 얼룩 전자 현미경 검사 법 방법: 시스템에 도전 하는 태 클을 위한 도구

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of ...

Measuring Trans-Plasma Membrane Electron Transport by 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 by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

C2C12 Myotubes에 의해 트랜스-플라즈마 막 전자 전송 측정

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

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Hsp33의 Redox 규제 보호자 활동을 정의 하 고 수소 중수소 교환 질량 분석을 사용 하 여 Hsp33에 구조적 변화를 매핑

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

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

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

세포 외 단백질 Microarray 기술은 낮은 친 화력 수용 체 Ligand 상호 작용의 높은 처리량 검색

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

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

분광학 그리고 스캐닝 전자 현미경 검사 법에 의해 섬유 소 혈전 구조에 β 아 밀 로이드 유도 비정상의 분석

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