这项工作在财政上得到了资助, 为特别促进研究从日本促进科学学会 (jsp) KAKENHI 授予数字 24000010, 17H04969 和 JP17J02602, 美国海军研究全球办公室 (N62909-17-1-2038)。Y.T. 是一个 jsp 研究研究员, 并通过该项目的领导研究生学校 (功绩) 支持的 jsp。

1. 在 ITO 电极上形成oneidensis MR-1 的单层生物膜 (图 1)
注: 为了防止电化学反应器与其他微生物的污染, 电化学反应器的所有介质、装置和元件应预先消毒。当使用oneidensis MR-1 单元格并构造电化学反应器时, 所有程序都应在干净的工作台上进行。
<ol><li>oneidensis MR-1 细胞的培养 注: 在以前报告的4的条件下, 在 ITO 电极上形成了一个oneidensis MR-1 的单层生物膜。<ol>
<li>要增长oneidensis MR-1 单元格, 请添加一个 s 的殖民地. oneidensis MR-1 在含有 Luria Bertani (磅) (20 克/升) 和 bacto 琼脂 (15 克/升) 的琼脂盘上生长, 在有氧条件下为30摄氏度的15毫升的 LB 培养基 (20 克/升), 在小时内以 24 rpm 摇晃。</li>
		<li>离心细胞悬浮在 6000 x g为10分钟, 并并用重悬在15毫升的被定义的培养基 (pH 7.8) 的结果细胞颗粒 (DM: NaHCO3 [2.5 克/升], CaCl2· 2H2O [0.08 克/升], NH4Cl [1.0 克/升], 氯化镁2·6H2O [0.2 克/升], 氯化钠 [10 克/升], 和 2-[4-(2-羟乙基) 1-哌嗪基] 磺酸 [HEPES; 7.2 克/升]), 辅以10毫米乳酸和0.5 克/升酵母萃取物作为碳源,以及 S . oneidensis 的跟踪元素 MR-1分别。</li>
		<li>进一步培养细胞悬浮有氧在30°c 为 12 h 与震动在 160 rpm 和离心机再在 6000 x g 为10分钟. 用 DM 介质将所合成的细胞颗粒洗净两次, 然后在 10 x g 的电化学前进行离心处理. 实验。</li>
	</ol>
</li>
	<li>三电极电化学反应器的构造 (图 1)<ol>
<li>在反应器底部放置一个 ITO 基体作为工作电极。</li>
		<li>随后, 插入一个玻璃圆筒 (直径2厘米) 和聚四氟乙烯 (PTFE) 盖子。然后将银/AgCl (氯化钾饱和) 和铂丝插入反应器中, 分别作为参考和反电极。 注: 为防止空气和溶液泄漏, 请在各组件之间插入丁基橡胶板。</li>
		<li>添加4.0 毫升 DM 补充10毫米乳酸和0.5 克/升酵母萃取物进入电化学反应器。</li>
		<li>在确认电化学反应器没有泄漏的情况下, 将氮气流入电化学反应器中20分钟, 以维持电化学反应器内的厌氧条件。 注: 为防止其他微生物污染, 应在气体流入电化学反应器之前对其进行过滤。</li>
		<li>将电化学反应器连接到恒电位仪, 并将 +0.4 V (与标准氢电极, 她) 应用于 ITO 电极, 使用外部水循环系统将电化学反应器的温度保持在30摄氏度。 注: 为了防止外部电场的影响, 电化学反应器应放置在法拉第笼中。</li>
	</ol>
</li>
	<li>oneidensis MR-1 单元格的电化学培养 (图 1和图 2)<ol>
<li>将步骤1.1 中获得的悬浮液的细胞密度调整为1.43 在 600 nm (OD600) 的光密度, 用 DM 补充10毫米乳酸和0.5 克/升酵母萃取物。 注意: 要获得正确的 od600, 请在调整 od600至1.43 之前, 将 od600 ≤0.8 的单元格悬浮应用于紫外-可见光光谱仪。</li>
		<li>使用注射器将0.3 毫升的电池悬浮液注入电化学反应器中: 反应器中的外径600变为0.1。 注意: 增加0.3 毫升细胞悬浮与 od600 = 1.43 入电化学反应器包含 4.0 mL 媒介结果4.3 毫升解答与 od600 = 0.1。当使用不同体积的其他反应器时, 需要计算细胞密度。</li>
		<li>继续潜在的应用在 +0.4 V (与她) 到 ITO 电极25小时。 注: 为在 ITO 电极上形成单层生物膜, 请确保所产生的电流与图 2中的偏差小于50%。</li>
	</ol>
</li>
</ol>2. 用新鲜 DM 培养基替换上清液, 10 毫米乳酸 (图 3)
<ol><li>停止潜在的应用, 并断开电化学反应器从恒电位仪和水循环系统。</li>
	<li>新鲜 DM 培养基替换上清液10毫米乳酸<ol>
<li>将氮气放入含有10毫米乳酸的 DM 的瓶子中, 从培养基中除去氧气。</li>
		<li>用注射器缓慢地将电化学反应器内的所有上清液取出, 在流动的氮气下 (图 3a, b)。 注: 为了避免氮气对 ITO 电极上的生物膜进行破坏, 气体应在液体表面上方流动。</li>
		<li>使用注射器添加4.0 毫升含有10毫米乳酸的新鲜 DM (图 3c)。 注意: 为了避免破坏 ITO 电极上的生物膜, 慢慢地将介质沿电化学反应器的壁上注入。为了保持生物膜湿润, 应在清除清液后立即添加培养基。在清除清液后1分钟内注射, 不会影响来自oneidensis MR-1 生物膜的当前生产。</li>
		<li>倾斜电化学反应器, 以除去附着在电化学反应器壁上的所有上清液。</li>
		<li>重复步骤 2.2. 2-2. 2.4 三次共计。</li>
	</ol>
</li>
	<li>停止气体流动, 再将电化学反应器连接到恒电位仪, 在 303 K 上应用 +0.4 V (vs 她) 到 ITO 电极上。</li>
</ol>3. 增加氘水来测量 EET 过程中的绢 (图 4)
<ol><li>确认来自oneidensis MR-1 的单层生物膜的当前生产是稳定的, 不会迅速增加。如果电流急剧增加, 请等到电流稳定在5% 以内, 增加10分钟。 注: 在 ito 电极上形成单层生物膜, 不受其他微生物菌株的污染, 经 rDNA 序列和 ITO 电极上生物膜的扫描电子显微图像证实, 如前所述4。为确定 EET 过程的速率限制, 监测添加穿梭电子介质的效果, 如100µM anthraquinone-1-sulfonate (α AQS)。有关详细信息, 请参阅代表结果和参考15一节。</li>
	<li>使用注射器将40µL 缺氧 50% (v/v) d2o 添加到电化学反应器中, 这样在反应器中浓度为 0.5% (v/v) d2o。 注: 为了防止 d2o 添加对生物膜的损坏, 请插入 d2o。</li>
	<li>等待当前稳定, 然后将 D2O 增加到 4.0% (v/v)。 注意: 要获取绢值 (在 D2o 和 H2o 的存在下当前生产的比率), 请检查同一容量的 H2O 加法对当前生产的影响。</li>
</ol>

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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>

作者没有什么可透露的。

与蛋白质电化学相比, 我们的全细胞电化学检测具有多种技术优势。蛋白质纯化需要多步骤的耗时程序, 但我们的全细胞方法在细胞培养后需要一天的自组织生物膜形成。为了实现 OM c-青旅和电极之间的稳定交互, 我们只需要对电极表面进行杀菌和清洗;它不需要电极修改来组织蛋白质的方向4,例如, S. oneidensis MR-1 本质上附着在电极上, 同时将 OM c-青旅定?...

在一个完整的细菌细胞中, 直接表征氧化还原蛋白的电化学技术最近出现, 自从发现了金属还原微生物菌株, 如S. oneidensis MR-1 或地杆菌能 sulfurreducens PCA,其中有外膜 c 型细胞色素复合体 (OM c-青旅) 暴露于细胞外部1,2,3,4,5。OM c-青旅介导从呼吸道链到位于 extracellularly 的固体基底的电子传输。此传输称为胞外电子传输 (EET)1,6 , 是新兴生物技术 (如微生物燃料电池6) 的关键过程。因此, 为了了解潜在的 EET 动力学和机制, 以及它与微生物生理学的联系, OM c-中青旅已使用全细胞电化学47, 结合显微术进行了研究。8,9, 光谱学10,11, 分子生物学2,4。相比之下, 研究 EET 相关的阳离子传输 (例如,质子) 对活细胞中 EET 动力学的影响的方法几乎没有建立, 尽管质子在细菌膜中的转运具有关键作用, 在信号、稳态和能源生产12,13,14。在本研究中, 我们描述了一个技术, 以检查质子传输对 EET 动力学的影响, 在S. oneidensis MR-1 细胞使用全细胞电化学测量, 这需要确定的速率限制步骤在微生物电流生产15。
一个直接的方法来评估质子传输对相关 EET 的贡献是氘动能同位素效应 (绢)。绢是可观察的作为电子转移动力学的变化在替换质子与氘离子, 代表质子传输对电子转移动力学的影响16。绢本身的理论已得到很好的建立使用电化学测量与纯化酶17。但是, 由于oneidensis中的当前生产 MR-1 来自多个、不同和波动的进程18, 因此不能简单地将 EET 标识为速率限制过程。为了观察质子传输过程与 EET 耦合的绢, 我们需要确认微生物电流的产生是通过 OM c-中青旅对电极的电子传输来限制的。为此, 在绢测量前, 我们在最佳 pH 值和温度条件下, 用含有高浓度乳酸的新鲜培养基取代上清液;这个替换服务两个角色: (1) 它提高了上游代谢过程的速率与 EET 相比, (2) 省略了在工作电极上从S. oneidensis MR-1 的单层生物膜上释放的上清液中的游泳细胞 (铟锡掺杂氧化物 (ITO) 电极)。所提供的详细协议旨在帮助新的从业者保持并确认 EET 过程是速率确定步骤。

<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&amp;Omega;/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, &amp;Phi;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 &lt;em&gt;S. oneidensis&lt;/em&gt; 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 (&amp;alpha;-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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Wako</td><td>191-01305</td><td>Used for defined medium (DM)</td></tr><tr><td>CaCl2 &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>031-00435</td><td>Used for DM</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Wako</td><td>011-03015</td><td>Used for DM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O</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&amp;times;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

在细菌外膜中嵌入的c型细胞色素复合物的直接电化学检测 (外膜c型细胞色素复合物;OM c-青旅) 最近出现作为一种新的全细胞分析方法来表征细菌电子运输从呼吸道链到细胞外部, 称为细胞外电子转运 (EET)。研究了 EET 反应过程中电子流的途径和动力学, 研究了与 EET 相关的阳离子输送的影响的全细胞电化学方法尚未建立。在本研究中, 本文介绍了通过 OM c-中青旅使用模型微生物 (希瓦氏菌 oneidensis MR-1) 检测 EET 上氘动力学同位素效应 (绢) 的生物化学技术的例子。如果 EET 通过 OM c-青旅作为微生物电流生产中的速率限制步骤, 则可以获得 EET 过程的绢。为此, 在添加 D2O 之前, 将上清液替换为含有足够数量的电子捐献者的新鲜介质, 以支持上游代谢反应的速率, 并从统一的浮游细胞中移除工作电极上的单层生物膜。此外, 还介绍了通过 OM c-青旅法确定微生物电流生产中 EET 速率限制步骤的替代方法。研究质子转运动力学的全细胞电化学检测技术可应用于其它活性微生物菌株。

25 h 的潜在应用在 +0.4 V (对她), 在 ITO 玻璃的工作电极上形成单层生物膜, 以前是通过扫描电子显微镜或共聚焦显微镜4确认的。在单层生物膜形成过程中, 从oneidensis MR-1 的当前生产的代表性时间过程显示在图 2中。尽管电流在每次测量中都有改变, 但如果单层生物膜均匀形成, 则产生的电流不会从图 2中的?...

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

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

在这里, 我们提出了一个全细胞电化学实验的协议, 以研究质子传输的贡献, 通过外膜细胞色素复合体在希瓦氏菌 oneidensis MR-1 的细胞外电子转运率。

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

electrochemical-detection-deuterium-kinetic-isotope-effect-on

Research

JoVE Journal

Biochemistry

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

9.9K 次观看.  University of Tokyo. 在这里, 我们提出了一个全细胞电化学实验的协议, 以研究质子传输的贡献, 通过外膜细胞色素复合体在希瓦氏菌 oneidensis MR-1 的细胞外电子转运率。

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

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

科研

环境科学

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.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

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

生物化学

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

生物工程

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 Myotubes 测量反式等离子体膜电子传输

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

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be critical for microbial energy production and persistence in many subsurface environments, especially those lacking soluble terminal electron acceptors. While EET-capable microbes have been successfully isolated from various environments, the diversity of bacteria capable of EET is still poorly understood, especially in difficult-to-sample, low energy or extreme environments, such as many subsurface ecosystems. Here, we describe an on-site electrochemical system to enrich EET-capable bacteria using an anode as a respiratory terminal electron acceptor. This anode is connected&#160;to a cathode capable of&#160;catalyzing abiotic oxygen reduction. Comparing this approach with electrocultivation methods that use a potentiostat for poising the electrode potential, the two-electrode system does not require an external power source. We present an example of our on-site enrichment utilized in an alkaline pond at the Cedars, a terrestrial serpentinization site in Northern California. Prior attempts to cultivate mineral reducing bacteria were unsuccessful, which is likely due to the low-biomass nature of this site and/or the low relative abundance of metal reducing microbes. Prior to implementing our two-electrode enrichment, we measured the vertical profile of dissolved oxygen concentration. This allowed us to place the carbon felt anode and platinum-electroplated carbon felt cathode at depths that would support anaerobic and aerobic processes, respectively. Following on-site incubation, we further enriched the anodic electrode in the laboratory and confirmed&#160;a distinct microbial community compared to the surface-attached or biofilm communities normally observed at the Cedars. This enrichment subsequently led to the isolation of the first electrogenic microbe from the Cedars. This method of on-site microbial enrichment has the potential to greatly enhance the isolation of EET-capable bacteria from low biomass or difficult to sample habitats.

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

自立电化学建立富集阳极 respiring 菌现场

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be ...

Characterizing Electron Transport through Living Biofilms

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under physiologically relevant conditions.1 These measurements are performed on living biofilms in aqueous medium using source and drain electrodes patterned on a glass surface in a specialized configuration referred to as an interdigitated electrode (IDA) array. A biofilm is grown that extends across the gap connecting the source and drain. Potentials are applied to the electrodes (ES and ED) generating a source-drain current (ISD) through the biofilm between the electrodes. The dependency of electrical conductivity on gate potential (the average of the source and drain potentials, EG = [ED + ES]/2) is determined by systematically changing the gate potential and measuring the resulting source-drain current. The dependency of conductivity on gate potential provides mechanistic information about the extracellular electron transport process underlying the electrical conductivity of the specific biofilm under investigation. The electrochemical gating measurement method described here is based directly on that used by M. S. Wrighton2,3 and colleagues and R. W. Murray4,5,6 and colleagues in the 1980&#39;s to investigate thin film conductive polymers.

A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

通过活生物膜表征电子传输

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

化学

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

原位萨尔和 TOF-SIMS希瓦氏菌 oneidensis MR1 生物膜的表征

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

<meta charset="utf8"></head><body>DHNA 对 植物乳植杆菌 EET 进行计时安培分析

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.

<meta charset="utf8"></head><body>用三电极、两室生物电化学系统表征乳酸菌介导的细胞外电子转移

Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.