Name: electrochemical detection of deuterium kinetic isotope effect on extracellular electron transport in shewanella oneidensis mr-1
Uploaded: 2018-04-16T14:30:00.000+00:00
Duration: 9 min
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

この作品の財政上支えられる補助金・特別推進研究の科学振興費助成番号 24000010、17 H 04969、日本社会からの JP17J02602、米国オフィスの海軍研究のグローバル (N62909-17-1-2038)。保は、日本学術振興会特別研究員、プログラムを通じて日本学術振興会での主要な大学院の学校 (メリット) をサポートします。

1. s. oneidensis氏-1 ITO 電極 (図 1) 上の単分子膜バイオ フィルムの形成
注: 他の微生物の電気化学リアクターの汚染を防ぐためには、すべてのメディア、実装、および電気化学リアクターのコンポーネント殺菌を行う事前に。ときにすべてのプロシージャをクリーン ベンチで行うべきs. oneidensis氏 1 セルを使用し、電気化学リアクターを構築します。
<ol><li>S. oneidensis氏 1 細胞の培養 注: s. oneidensis氏 1 の単分子膜バイオ フィルムが形成された ITO 電極条件を以下報告以前4。<ol>
<li>S. oneidensis氏 1 細胞を成長するには、 s. oneidensis氏-1 ルリア ベルターニカミラ (LB) (20 g/L) を有し、15 mL の LB 培地 (20 g/L) 160 rpm で振とうしながら好気性条件で 24 h の 30 ° c になる寒天 (15 g/L) 寒天プレート上に成長のコロニーを追加します。</li>
		<li>6,000 × g , 10 分間の細胞懸濁液を遠心し、15 mL の培地 (pH 7.8) で得られた細胞ペレットを再懸濁します (DM: NaHCO3 【 2.5 g/L CaCl2·2H2O [0.08 g/L]、NH4Cl 【 1.0 g/L MgCl2·6 H2O 【 0.2 g/L 塩化ナトリウム 10 g/L と 2-[4-(2-hydroxyethyl)-1-piperazinyl] [HEPES; 7.2 g/L] ethanesulfonic 酸) 10 mM 乳酸と 0.5 g/L 酵母エキス、炭素源として補われた、 s. oneidensis氏 - 1 微量元素、それぞれ。</li>
		<li>さらに 160 rpm で振とうしながら 12 h 30 ° C で好気性細胞懸濁液を育成、遠心力場再び 6,000 × g 10 分洗浄で DM 媒体で 2 回結果細胞ペレットの電気化学的前に 6,000 × g で 10 分間遠心分離によって実験。</li>
	</ol>
</li>
	<li>3 電極電気化学リアクター (図 1) の建設<ol>
<li>原子炉の下部に作用電極として ITO 基板を置きます。</li>
		<li>その後、ガラス シリンダー (直径 2 cm)、ポリテトラフルオロ エチレン (PTFE) カバーを挿入します。銀/塩化銀 (飽和 KCl) と白金線に挿入原子炉参照とカウンター電極としてそれぞれ。 注: 空気およびソリューションの漏れを防ぐためには、各コンポーネント間にブチルゴム シートを挿入します。</li>
		<li>10 mM 乳酸を添加した DM の 4.0 mL と 0.5 g/L 酵母エキス電気化学リアクターに追加します。</li>
		<li>電気化学リアクターからの漏れがないことを確認した後、窒素ガスを電気化学リアクター電気化学リアクター内の嫌気的条件を維持するために 20 分以上に流れ込みます。 注: 他の微生物による汚染を防ぐためには、ガスがフィルターされ、電気化学リアクターに流れる前に、。</li>
		<li>電気化学リアクターを電気化学測定装置に接続し、0.4 V を適用 (標準的な水素の電極対彼女) ITO 電極に外部の水循環システムを使用して 30 の ° C で電気化学リアクターの温度を維持します。 注: 外部電界の影響を防ぐためには、電気化学リアクターはファラデーケージの中に配置する必要があります。</li>
	</ol>
</li>
	<li>(図 1および図 2) s. oneidensis氏 1 セルの電気化学的栽培<ol>
<li>手順 1.1 600 1.43 の光学密度で得られた懸濁液の細胞密度を調整 10 mM 乳酸と 0.5 g/L 酵母によって補われる DM の nm (外径600) を抽出します。 注: 正しい外径600を得るためには、OD600 ≦ 0.8 細胞懸濁液を調整 1.43 に OD600前に紫外可視分光光度計に適用します。</li>
		<li>注射器を使って注入ポートを介して電気化学リアクターに細胞懸濁液 0.3 mL を追加: 原子炉で OD600は 0.1 に変更します。 注: 外径600 0.3 mL 細胞懸濁液の添加外径600ソリューション 4.3 ml 4.0 mL 中結果を含む電気化学リアクターに 1.43 を = = 0.1。別のボリュームとその他の原子炉を使用している場合、細胞密度の計算が必要です。</li>
		<li>25 h ITO 電極に (彼女) と 0.4 V で潜在的なアプリケーションを続行します。 注: ITO 電極上の単分子膜バイオ フィルムの形成、作り出された電流を示す偏差が図 2の 50% 未満の場合ことを確認します。</li>
	</ol>
</li>
</ol>2. 10 mM 乳酸 (図 3) と新鮮な DM 媒体と上澄みの交換
<ol><li>潜在的なアプリケーションを停止し、電気化学リアクターをポテンショスタットと水循環システムから外します。</li>
	<li>10 ミリメートルと新鮮な DM 媒体と上澄みの交換乳酸します。<ol>
<li>10 mM 乳酸 20 分以上で DM を含む媒体から酸素を取り除くボトルに窒素ガスを流れ。</li>
		<li>ゆっくりと流れる下、注射器を使用して電気化学反応器内培養上清を削除 (図 3 a, b) の窒素ガス。 注: 窒素ガスによる ITO 電極上のバイオ フィルムを破損しないよう、ガスは液体表面上フローする必要があります。</li>
		<li>注射器 (図 3 c) を使用して 10 mM 乳酸を含む新鮮な DM の 4.0 mL を追加します。 注: ITO 電極上のバイオ フィルムを破損しないよう、電気化学リアクターの壁に沿って媒体をゆっくりと挿入します。濡れているバイオ フィルムを保つためには、上清を除去した後すぐに媒体を追加する必要があります。上清を除去した後の中の最大 1 分の注入は、 s. oneidensis氏 1 のバイオから現在の生産には影響しません。</li>
		<li>電気化学リアクターの壁に付着した上清のすべてを削除する電気化学リアクターを傾けます。</li>
		<li>合計で 3 回の手順 2.2.2-2.2.4 を繰り返します。</li>
	</ol>
</li>
	<li>ガスの流れを停止し、電気化学リアクターを 303 K で ITO 電極に 0.4 V (彼女) の対を適用するもう一度、ポテンシオスタットに接続</li>
</ol>3. EET プロセス (図 4) に KIE を測定する重水素水の添加
<ol><li>S. oneidensis氏 1 の単分子膜バイオ フィルムから現在の生産は安定している、急速に増加しませんを確認します。電流が急激に増加、現在安定して ± 5% 増加 10 分以上までを待ちます。 注:4を既に報告されている、他の微生物の系統の混入しない ITO 電極上の単分子膜バイオ フィルムの形成が rdna 塩基配列と ITO 電極上のバイオ フィルムの走査型電子顕微鏡像で確認をしました。EET プロセスによる速度制限の確認、往復の電子メディエーター 100 μ M アントラキノン-1-スルホン酸 (α-AQS) などの追加の効果を監視します。代表結果のセクションを参照してください、詳細については、15を参照します。</li>
	<li>濃度は 0.5% (v/v) D2O 原子炉のような注射器を使用して電気化学リアクターに無酸素 50% (v/v) D2O の 40 μ L を追加します。 注: D2O 添加によるバイオ フィルムへの損傷を防ぐため、注入 D2O drop-wise。</li>
	<li>安定し、現在待って、その後 D2O 4.0% (v/v) を追加します。 注: KIE 値 (D2O と H2O の存在下で現在の生産に対する比率) を得るためには、同じ現在の生産における H2O 添加量の効果を確認します。</li>
</ol>

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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>

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我々 の全細胞の電気化学的分析タンパク質電気化学と比較していくつかの技術的な利点があります。蛋白質の浄化には、多段階の時間のかかる手続きが必要ですが、本全体セル手法で自己組織化バイオ フィルム形成細胞培養後 1 日は。OM cの安定した相互作用を達成するために - 假と電極、必要がありますのみ殺菌と電極表面のクリーニングOM cを定位しながら、電極上にアタッ...

S. oneidensis氏 1、 Geobacter sulfurreducens 、PCA など、金属還元微生物菌株が発見されて以来そのままの細菌の細胞の酸化還元タンパク質を直接特徴付けるため電気化学的手法が近年します。外膜 c 型チトクロム複合体 (OM c 假) セル外観1,2,3,4、5の露出があります。OM c- 假は、呼吸鎖から細胞外に位置する固体基板上への電子輸送を仲介します。このトランスポートは、細胞外の電子輸送 (EET)1,6と呼ばれ、新興のバイオ テクノロジー、微生物燃料電池6などの重要なプロセスです。したがって、基になる EET の速度論と機構、および微生物生理学へのリンクを理解する OM c -假を用いて調べた細胞電気化学4、7顕微鏡と組み合わせて8,9、分光10、11、および分子生物学2,4。対照的に、EET 関連する陽イオン輸送など陽子 EET 細胞内動態の影響を調査する方法確立されているやっとのこと重要な役割を持つ細菌の膜を渡るプロトン輸送にもかかわらずシグナリング、恒常性とエネルギー生産12,13,14。本研究で、細胞電気化学測定法を使用しての率制限ステップの id を必要とするs. oneidensis氏 1 セルの EET 動力学に関するプロトン輸送の影響を調査する手法について述べる微生物現在生産15。
関連付けられた EET のプロトン輸送の貢献を評価する直接方法の 1 つは重水素同位体効果 (キエです)。KIE 電子転送速度16プロトン輸送の影響を表す重水素イオン、プロトンの交換時に電子伝達速度の変化として観察可能であります。KIE 自体の理論定着させてきた電気化学測定法を用いた精製酵素17。しかし、以来、 s. oneidensis氏-1 の現在の生産の結果、複数の多様なと変動プロセス18から、レート制限プロセスとして EET を識別できない単に 1 つ。EET と相まってプロトン輸送過程の KIE を観察する我々 は OM cを介して電子輸送によって微生物の現在の生産が制限されることを確認する必要があります - 電極に假。この目的のため最適 pH と KIE 測定前に温度条件を電子供与体として乳酸の高濃度を含む新鮮な培地と培養上清中のソリューションを置き換えこの交換は、2 つの役割を提供しています: (1) これに EET と比較して上流の代謝プロセスのレートを強化、(2) 作業電極 ( s. oneidensis氏 1 の単分子膜バイオ フィルムから発売清スイミング セルの省略インジウム スズ添加酸化物 (ITO) 電極)。提示の詳しいプロトコルを新しい実務を維持し、EET プロセスが、律速段階であることを確認します。

<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

直接cの電気化学的検出-細菌の外膜に埋め込まれているシトクロム複合体を入力 (外膜c-シトクロム複合体; を入力OM c- 假) が最近セル外部へ呼吸の鎖からの細菌の電子輸送を特徴付ける新規細胞分析法として登場、細胞外の電子輸送 (EET) と呼ばれます。経路と EET 反応の間に電子流の動態を調べた、一方 EET に関連付けられている陽イオン輸送の影響を調べる全細胞の電気化学的手法はまだ確立されていません。本研究では、重水素同位体効果 (キエ) EET の OM cを調べる生化学的手法の例 - 假モデル微生物、シェワネラ oneidensis氏-1, を使用して、説明します。OM cを介して EET - 假微生物の現在の生産の率制限ステップとして機能する場合、EET プロセスに KIE を取得できます。そのため、D2O は、加算の前に上澄みが上流の代謝反応の速度をサポートし、ユニフォームから浮遊性のセルを削除する電子供与体の十分な量が含まれている新鮮なメディアに置き換えられました作用電極上の単分子膜バイオ フィルム。レート制限を確認する方法は、微生物の現在の生産のステップ - 假 OM cを介して EET として述べる。プロトン輸送の動力学を調査するため細胞の電気化学的アッセイの我々 の手法は、他の電気活性微生物系統に適用できます。

(彼女) と 0.4 V で潜在的なアプリケーションの 25 時間後単分子膜バイオ フィルムは ITO ガラス、走査型電子顕微鏡や共焦点顕微鏡4で確認された以前の作用電極に形成されました。単分子膜バイオ フィルムの形成の間にs. oneidensis氏 1 から現在の生産の代表的な時間コースを図 2に示します。現在の生産は現在の間隔で変更...

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

電気化学検出の重水素速度論的同位体効果シェワネラ oneidensis氏 1 の細胞外電子輸送

ここで全細胞外膜シトクロムシェワネラ oneidensis氏-1 複合体を介して細胞外の電子輸送の速度へのプロトン輸送の貢献を研究する電気化学実験のプロトコルを提案する.

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

10.0K ビュー.  University of Tokyo. ここで全細胞外膜シトクロムシェワネラ oneidensis氏-1 複合体を介して細胞外の電子輸送の速度へのプロトン輸送の貢献を研究する電気化学実験のプロトコルを提案する.

ビデオ: 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 ...

環境学

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.

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

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

生化学

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.

電気化学センサを用いた細菌検出＆識別

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

生物工学

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 細胞によるトランス プラズマ膜電子輸送特性の測定

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).

敷地内に陽極 respiring 細菌を豊かに自立の電気化学的セットアップ

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.

生きているバイオ フィルムにおける電子輸送特性評価

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.

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオ フィルムの特性

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.

ペプチド生合成酵素を調べた水素重水素交換質量分析(HDX-MS)プラットフォーム

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を用いた Lactiplantibacillus plantarum におけるEETのクロノアンペロメトリー解析

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>乳酸菌における媒介細胞外電子移動の特性評価 3電極2チャンバー生体電気化学システムによる

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.

電気化学検出の重水素速度論的同位体効果シェワネラ oneidensis氏 1 の細胞外電子輸送

要約

さらに動画を探す

この動画の章

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

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

電気化学センサを用いた細菌検出＆識別

C2C12 細胞によるトランスプラズマ膜電子輸送特性の測定

敷地内に陽極 respiring 細菌を豊かに自立の電気化学的セットアップ

生きているバイオフィルムにおける電子輸送特性評価

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオフィルムの特性

ペプチド生合成酵素を調べた水素重水素交換質量分析(HDX-MS)プラットフォーム

乳酸菌における媒介細胞外電子移動の特性評価 3電極2チャンバー生体電気化学システムによる

乳酸菌における媒介細胞外電子移動の特性評価 3電極2チャンバー生体電気化学システムによる

電気化学検出の重水素速度論的同位体効果シェワネラ oneidensis氏 1 の細胞外電子輸送

要約

さらに動画を探す

この動画の章

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

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

電気化学センサを用いた細菌検出＆識別

C2C12 細胞によるトランス プラズマ膜電子輸送特性の測定

敷地内に陽極 respiring 細菌を豊かに自立の電気化学的セットアップ

生きているバイオ フィルムにおける電子輸送特性評価

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオ フィルムの特性

ペプチド生合成酵素を調べた水素重水素交換質量分析(HDX-MS)プラットフォーム

乳酸菌における媒介細胞外電子移動の特性評価 3電極2チャンバー生体電気化学システムによる

乳酸菌における媒介細胞外電子移動の特性評価 3電極2チャンバー生体電気化学システムによる

C2C12 細胞によるトランスプラズマ膜電子輸送特性の測定

生きているバイオフィルムにおける電子輸送特性評価

その場でサルヴィ、TOF-SIMSシェワネラ oneidensis MR1 バイオフィルムの特性