このビデオの記事で行った実験国際遺伝子改変マシン競争9のために開発されました。著者はIGEMのチームメンバーにルークBergwerff、ピーテルTMバンBoheemen、Jelmerクノッセン、ヒューゴ·F Cuetoロハスとするためラモンファンデルファルクに感謝したいと思います研究の援助。我々は有用な議論ハン·デ·Winde、ステファンデコックとEsengülYILDIRIMに感謝し、この研究をホスティング。この作品は、バイオテクノロジーのデルフト工科大学部、デルフトインフォマティクス研究室、Bionanoscienceのデルフト工科大学学科、オイルサンドリーダーシップイニシアティブ（OSLI）、スタッドstudentenuitzendbureau、オランダゲノミクスイニシアティブ、Kluyverセンター、Nederlandse Vereniging Biotechnologische（開発元：Stichtingバイオオランダ）によってサポートされていました、DSM、GENEART、グライナーバイオ1とジェネンコア。

1。 BioBrickアセンブリ<ol><li>標準生物学的部分のレジストリからBioBricksはIGEM本部によって提供されています。既存BioBricksから新しいBioBrickを構築するために、アクセプター部分のドナー部分の下流に位置決めするための酵素EcoRIとSpeIでドナーBioBrick（最大1.0μg）を消化。アクセプター部の上流ドナー部分を位置決めするためのXbaIとPstIでダイジェスト。ドナーのバックボーンにカット第三適切な制限酵素を追加します。サプライヤー（最終濃度1X）によると、適切な緩衝液20〜25 mlの総体積で消化を行う。制限酵素のた​​めの5単位/μgのDNAを使用しています。 </li><li> EcoRIおよびXbaIまたはSpeIとPstIでどちらかとアクセプタBioBrickを消化。 </li><li> 37℃で1時間（少なくとも）のために消化をインキュベート℃に制限エンドヌクレアーゼは、熱によって不活性化、80℃インキュベーションまもなく10分間遠心℃で。 </li><li>消化BioBrickを連結一緒にパーツ（ドナーとアクセプター）。 XbaIとSpeIでは、互換性のDNA末端を生成するので、混合型のサイトは、4つの標準的な制限部位に隣接、新しい &quot;複合&quot; BioBrickに起因するいかなる制限酵素で切断することはできませんが作成されます。ライゲーション混合物で最終DNA濃度は、好ましくは約100 ng /μlに。 T4のライゲーションバッファー（最終濃度1x）とT4リガーゼ（1単位/μgのDNA）を10〜15 mLの全量ライゲーション反応を行う。 </li><li>少なくとも3時間16℃でライゲーション混合物をインキュベートする。 </li><li>ライゲーションミックスの年頃の半分を使った変換反応を行う。 </li><li>シーケンスと正しいことがBioBrickを確認します。 </li><li>合成されたDNAから新しいBioBrickを構築するために、Eの最適な発現のために合成するための遺伝子を変更JCatウェブツール（による大腸菌 <a href="http://www.jcat.de/" target="_blank">http://www.jcat.de/</a> ）。 Bの要件に合わせてさらに変更を加え<html> ioBrick標準。この標準化は、すべてのBioBrickがプレフィックスが先行し、接尾辞が続く目的のDNA配列で構成されていることを意味します。接頭辞と接尾辞は、残りのプラスミド配列には存在しない事前定義された制限部位を含む配列である。これらの標準的な制限部位は、インターチェンジと9柔軟BioBrickインサートを延長することが可能になる。接頭辞と接尾辞は、以下の配列を持っている： </li></ol><table border="1"><tbody><tr><td> 名前 </td><td> シーケンス </td><td> コメント </td></tr><tr><td>接頭辞</td><td> 5 &#39;GAATTCGCGGCCGCTTCTAG3&#39; </td><td></td></tr><tr><td></td><td> 5 &#39;GAATTCGCGGCCGCTTCTAGAG 3&#39; </td><td>次の部分は、コード配列または &quot;ATG&quot;で始まる任意の一部である場合</td></tr><tr><td>サフィックス</td><td> 5 &#39;TACTAGTAGCGGCCGCTGCAG 3&#39; </td><td></td></tr></tbody></table>Vivoで ove_title &quot;&gt; 2。アルカン変換一休み細胞アッセイ、 このアッセイは、藤井ら （2004）によって記載された方法に基づいて行われた。 <ol><li>文化、E.アルカン水酸化酵素系（発現している大腸菌細胞<a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a>空ベクター（簿価）と細胞<a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002を</a>一晩適切な抗生物質を5 mlのLB培地中で）を。 </li><li>新鮮なLBの50ml（抗生物質を含む）に一晩培養液500μlを移すと、細胞濁度が600nmで0.3のOD（光学密度）に達するまでインキュベートする。 </li><li> 4,000 rpmで10分間遠心し、5mlの0.1Mリン酸緩衝液（pH7.4）でペレットを再懸濁します。 </li><li> 4,000 rpmで10分間遠心分離し、再び今のE2の塩および0.66パーセントv / vグリセロール（窒素deficienを含む5mlの0.1Mリン酸緩衝液中でペレットを再懸濁トン培地）。 </li><li>細胞濁度（OD 600）を測定します 。 </li><li> 25ミリリットルクローズドキャップガラスフラスコおよび無細胞コントロールで6ミリリットル（E2塩+ 0.66％（v / v）のグリセロール）の細胞混合物のアリコートを準備します。 </li><li>各フラスコにアルカンの100ナノモルを追加します。 </li><li> 24時間（より高いレートが得られたときに短縮）37℃で混合物をインキュベートする。 </li><li>インキュベーション後、OD 600を測定します 。 </li><li>酢酸エチルで培地中の炭化水素を抽出し、ガスクロマトグラフィー（プロトコル3を参照）により、細胞培養における炭化水素濃度を測定します。 </li><li>各フラスコ内の全バイオマス存在する（乾燥重量のOD 600に変換）によって変換されたアルカンの合計額で除してバイオマスの単位あたりの劣化を計算します。実験期間で結果を分割する単位時間当たり、単位当たりのバイオマス分解活性が得られます。平均は3個々の実行から取得されます。 </li></ol> 3。アルカン変換酵素アッセイでは 、ビトロこのアッセイは、Li ら （2008）により記載された方法に本質的に従って行った。 <ol><li>文化、E. LADA遺伝子（発現している大腸菌細胞<a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a> ）と空ベクター（担持する細胞<a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002を</a>一晩適切な抗生物質を50mlのLB培地中で）を。 </li><li>新鮮なLBの50ml（抗生物質を含む）に一晩培養液500μlを移すと、細胞濁度が600nmで0.6の光学密度に達するまで培養する。 </li><li> 4000 RPM（4℃）で10分間遠心分離し、50mMトリス緩衝液5mlにペレットを再懸濁します。 </li><li> 4の出力制御を40％のデューティサイクルで超音波洗浄します（細胞破砕、LA Biosystems社）細胞は、全期間にわたって氷の上で解決策を保つ。 </li><li> 4＆dで4,000 rpmで5分間遠心し、得られた混合物を例えば、C細胞破片を除去する。新鮮なバイアルに上清を移してください。 </li><li>ブラッドフォードアッセイにより細胞抽出物の総タンパク質濃度を決定します。注：バックグラウンドの増加および/またはタンパク質の損失を防ぐために、ガラスバイアルを使用します。 </li><li> 0.1パーセントv / vのアルカンおよび50mM Tris-HCl緩衝液を含む100mlの混合物を準備します。 </li><li> 5分間100℃で混合物を加熱する。注：のみ沸点の高い中型の長鎖アルカンに対してこの手順を実行します。 （ 例えば、C 16：287℃）。 </li><li>均質、粘性の混合物が得られるまでアルカンの最適な溶解性を達成するためには、まだ温かいうちに1分間超音波洗浄します。 </li><li> NADHの1mm、1mMのFMN、1mMのMgSO 4および0.01 V / VのTriton X-100を追加します。 </li><li> 25ミリリットルクローズドキャップフラスコに6 mlのアリコートを準備します。 </li><li>細胞抽出物（ブラッドフォードアッセイ、5 mgタンパク質/ lの最終濃度に依存します）の適切な量を追加します。無タンパク質制御を準備します。 </li><li> 60℃でインキュベート（最適なeについて24時間nzymatic活動）。 </li><li>酢酸エチルで培地中の炭化水素を抽出し、ガスクロマトグラフィー（プロトコル3を参照）により、細胞培養における炭化水素濃度を測定します。 </li><li>各フラスコ（ブラッドフォード検量線による）に加え全タンパク質によって変換されたアルカンの合計額で除してバイオマスの単位あたりの劣化を計算します。さらに実験期間で割ると、単位時間あたりの細胞タンパク質の単位当たりの分解活性が得られます。平均は3個々の実行から取得されます。 </li></ol> 4。酢酸エチルの炭化水素抽出と濃度測定<ol><li>アルカンは6ミリリットル実験溶液中に2.5ミリリットルの酢酸エチル（無極性solvant）を加えることによって、水溶液から抽出されます。内部標準は、0.1％（v / v）の濃度で溶媒に添加される。標準（ 例えばシクロデカン）興味のあるピークの予想範囲に応じて変化した。 </li><li>オプション：追加トリトン水性混合物のX-100および適切な二相系を得るために4,000 rpmで10分間サンプルを遠心分離する。 </li><li>ボルテックス5秒（1,500 rpm）と別々の二つの相になるまで室温でインキュベートするための混合物。 </li><li>有機層（上部）の最大量を削除し、無水硫酸マグネシウムを用いて溶媒を乾燥させてください。 </li><li>ろ過によりMgSO 4を取り外します（0.2μm）と測定のためのガスクロマトグラフバイアルにろ液を転送、または-20℃で保存する</li><li> CP-SIL 5CB列（長さ= 5メートル）を使用してガスクロマトグラフィーを用いて濃度を測定します。 （1:10、230℃）、スプリットモードではサンプルの10μlを注入します。 1ml /分（ヘリウム）にカラムガス流量を設定します。以下のオーブン温度プログラムは使用されています： <table border="1"><tbody><tr><td> 率 </td><td> 温度[°C] </td><td> 時間[分] </td></tr><tr><td> 0 </td><td> 50 </td><td> 7.5 </td></tr><tr><td> 50 </td><td> 90 </td><td> 1.0 </td></tr><tr><td> 50 </td><td> 110 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 130 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 145 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 160 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 170 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 185 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 210 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 250 </td><td> 2.0 </td></tr><tr><td> 50 </td><td> 320 </td><td> 2.0 </td></tr></tbody></table></li><li>ピークを積分し、内部標準に対する濃度を修正します。 </li></ol> 5。アルコール/アルデヒド脱水素酵素活性アッセイこのアッセイは、加藤らの方法によれば、基本的に行った。（2010）。 <ol><li>文化、E. ADH（発現している大腸菌細胞<a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a> ）またはALDH（ <a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a> ）遺伝子と空ベクター（担持する細胞<a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002を</a>一晩適切な抗生物質を50mlのLB培地中で）を。 </li><li>新鮮なLBの50ml（抗生物質を含む）に一晩培養液500μlを移すと、細胞濁度が600nmで0.6の光学密度に達するまで培養する。 </li><li> 4℃、4,000 rpmで10分間遠心分離し、50mMトリス緩衝液5mlにペレットを再懸濁します。 </li><li> 4の出力制御（超音波処理中に氷の上で解決策を保つ）と40％のデューティ·サイクルで細胞溶液を超音波洗浄します。 </li><li> °Cは、細胞破片を除去するために4℃、4,000 rpmで5分間、得られた混合物を遠心分離します。 </li><li>に決定Bradfordアッセイ（：結合タンパク質を防止するために、ガラスバイアルを使用注）により細胞抽出物のタンパク質濃度TAL。 </li><li>各ウェル中のNAD（最終濃度1mM、pH 9.5）で含む180μlの57 mMグリシン緩衝液を用いて、96ウェルプレートをセットします。 </li><li>ウェルにテストされるアルコール（アルデヒド）の5μlを添加する。注：ヒート長鎖アルコールアッセイの開始前の液体を持っている。各アルコール（アルデヒド）のための基質を含まない対照（陰性コントロール）を追加する必要があります。加えて、細胞抽出せずにバッファと基板との混合物を含有するブランクを準備します。 </li><li>予熱37℃で15分間プレートリーダー（TecanマゼランV7.0）のプレート°C系の平衡化を可能にする。 </li><li>細胞抽出物（ブラッドフォードアッセイ、5 mgタンパク質/ lの最終濃度に依存します）の適切な量を追加します。無タンパク質制御を準備します。 </li><li> 340 nmの波長で37℃で1時間2〜3分ごと℃&lt;を分光光度計を用いてNADH産生を測定/ LI&gt; <li> OD（340 nm）の斜面からのNADHの​​生産率を計算します。アカウントへの光路長と6220 M -1 cm -1のNADHの吸光係数を取る。細胞抽出物（U / mgの全細胞タンパク質）における脱水素酵素反応の活性を発現するために追加されたタンパク質の総量によってNADH生産速度を割ります。平均値と3は独立して動作しますから標準偏差を計算します。 </li></ol> 6。 pCaiFキャラクタリゼーション<ol><li>文化、E. pCaiF-GFPコンストラクト（発現している大腸菌細胞<a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a> ）と単独のプロモーター（担持する細胞<a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326を</a>適切な抗生物質を一晩5mlのLB培地で一晩）。 </li><li>一晩培養した培養液50μlと、10g / lのグルコースおよび抗生物質を含む5mlのM9を接種し、一晩増殖。 </li&gt; <li>新鮮なM9 with10 g / lでインキュベートし、5mlの中に一晩伸長のサブカルチャー50μlの細胞濁度は、600nmで0.2の光学密度に達するまで。 </li><li>各ウェルにテストするための炭素源の所望の量を含有する新鮮なM9培地100μlの96ウェルプレートをセットします。統計的評価のために3回の実験を行い、それぞれのネガティブコントロール（野生型大腸菌 K12）を追加します。 </li><li>井戸を含む培地に一晩培養液5μlを添加する。 </li><li>成長曲線（OD 600）とプレートリーダーを用いてGFP蛍光（485 nm励起と520発光）37℃で18時間毎に10分°Cの定数と振とうして測定します。 </li><li>成長率は、それぞれの測定値から特定のGFP量を計算し、コントロールと比較します。平均値と少なくとも3つの独立した実験の標準偏差を計算します。 </li></ol> 7。許容アッセイ<ol><li>文化PhPFDαおよびβ遺伝子（発現する大腸菌 <a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a> 5 mlのLB培地と適切な抗生物質で一晩）、E. LADA遺伝子（発現している大腸菌は <a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a> ）ネガティブコントロールとして使用されます。 </li><li>新鮮なLBの5ミリリットル（抗生物質）とインキュベート中に一晩伸長のサブカルチャー10μlの細胞濁度は、600nmで0.3から0.4の光学密度に達するまで。 </li><li> 0.1のOD 600に到達するまで、新鮮な培地で培養液を希釈します。 </li><li>適切な抗生物質と三重で毒性化合物（ 例えば、0、4、8、n-ヘキサンの10％）の適切な最終濃度を含む180μlのM9培地で96ウェルプレートをセットします。アルカン-水混合物は、二相系につながる可能性があるので、それはプレート上の適切なコントロール（ 例えば dを持つことが不可欠であるifferent株とそれぞれのブランク実験）。 </li><li>ウェルに培養液20μl（コントロール）を追加します。 </li><li>一定振盪しながらプレートリーダーを37℃で24時間毎に10分°Cを使ってバイオマス濃度（OD 600）を測定します 。 </li><li>異なる薬剤濃度については、それぞれの測定値からの成長率を計算し、陰性対照と比較します。平均値と、少なくとも3つの独立した実行の標準偏差を計算します。 </li></ol> 8。同族体の相互作用のマッピング<ol><li>アプリケーションHIMは、STRINGデータベース（学術的な使用のため無料）20を稼動しているPostgreSQLサーバ上のタンパク質のクエリを実行するために開発されました。同族体の相互作用マッピングアプリケーションはSTRINGデータベースを使用して、元の宿主生物において作用するタンパク質を識別します。それぞれの相互作用遺伝子の配列は、標的生物の相同遺伝子を見つけるために、BLAST検索で使用されています。 Cytoscape 4 </sまで&gt;は、マッピングの結果を可視化するために使用されています。 ：HIMソフトウェアツールは次の場所にダウンロードすることができます<a href="https://github.com/jcnossen/InteractionHomologMapping" target="_blank">https://github.com/jcnossen/InteractionHomologMapping。</a> </li><li>マッピングを実行するには、（1）BioBrick IDを入力すると、アプリケーションは自動的に標準生物パーツ9、または（2）を入力（ペースト）、アプリケーション内の配列データのレジストリから一部のシーケンスデータをダウンロードします。 </li><li>入力したアミノ酸配列のSTRINGプロテインIDを見つけるために、文字列データベースのWebサイトを使用しています。 </li><li>高い相同性を持つタンパク質は、BLASTを用いて決定される。その後、アプリケーションは、宿主生物におけるホモログ（ 例えば大腸菌 ）のソース生物と検索で既知の各相互作用タンパク質を示しています。 </li><li>テキストまたはCytoscapeに結果の推定上の相互作用リストをエクスポートします。 </li></ol>

<ol>
	<li>Allen, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+water+treatment+in+Canada's+oil+sands+industry:+I:+Target+pollutants+and+treatment+objectives.">Process water treatment in Canada's oil sands industry: I: Target pollutants and treatment objectives.</a> J. Environ. Eng. Sci. 7, 123-138 (2008).</li><li>Alon, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Systems Biology: Design Principles of Biological Circuits. , (2007).</li><li>Center, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Understanding Oil Spills and Oil Spill Response. , (1999).</li><li>Eichler, K., Buchet, A., Lemke, R., Kleber, H. P., Mandrand-Berthelot, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+the+caiF+gene+encoding+a+potential+transcriptional+activator+of+carnitine+metabolism+in+Escherichia+coli.">Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli.</a> J. Bacteriol. 178, 1248-1257 (1995).</li><li>Feng, L., Wang, W., Cheng, J., Ren, Y., Zhao, G., Gao, C., Tang, Y., Liu, X., Han, W., Peng, X., 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=Genome+and+proteome+of+long-chain+alkane+degrading+Geobacillus+thermodenitrificans+NG80-2+isolated+from+a+deep-subsurface+oil+reservoir.">Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (13), 5602-5607 (2007).</li><li>Fujii, T., Narikawa, T., Takeda, K., Kato, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotransformation+of+various+alkanes+using+the+Escherichia+coli+expressing+an+alkane+hydroxylase+system+from+Gordonia+sp.+TF6.">Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6.</a> Biosci. Biotechnol. Biochem. 68 (10), 2171-2177 (2004).</li><li>Kato, T., Miyanaga, A., Haruki, M., Imanaka, T., Morikawa, M., Gene Kanaya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cloning+of+an+alcohol+dehydrogenase+from+thermophilic+alkane-degrading+Bacillus+thermoleovorans+B23.">cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23.</a> J. Biosci. Bioeng. 91 (1), 100-102 (2001).</li><li>Kato, T., Miyanaga, A., Kanaya, S., Morikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+cloning+and+characterization+of+an+aldehyde+dehydrogenase+from+long-chain+alkane-degrading+Geobacillus+thermoleovorans+B23.">Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23.</a> Extremophiles. 14, 33-39 (2010).</li><li>Kieboom, J., De Bont, J. a. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial Stress Responses. , (2000).</li><li>Kotte, O., Zaugg, J. B., Heinemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adaptation+through+distributed+sensing+of+metabolic+fluxes.">Bacterial adaptation through distributed sensing of metabolic fluxes.</a> Mol Syst Biol. 6, 355 (2010).</li><li>Kremling, A., Bettenbrock, K., Gilles, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+global+control+of+Escherichia+coli+carbohydrate+uptake.">Analysis of global control of Escherichia coli carbohydrate uptake.</a> BMC Syst. Biol. 1, (2007).</li><li>Li, L., Liu, X., Yang, W., Xu, F., Wang, W., Feng, L., Bartlam, M., Wang, L., Rao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+long-chain+alkane+monooxygenase+(LadA)+in+complex+with+coenzyme+FMN:+unveiling+the+long-chain+alkane+hydroxylase.">Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase.</a> J. Mol. Biol. 376 (2), 453-465 (2008).</li><li>Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S. O., Neubauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+maximum+specific+uptake+capacities+for+glucose+and+oxygen+in+glucose-limited+fed-batch+cultivations+of+Escherichia+coli.">Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli.</a> Biotechnol. Bioeng. 73 (5), 347-357 (2001).</li><li>Okochi, M., Kanie, K., Kurimoto, M., Yohda, M., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+prefoldin+from+the+hyperthermophilic+archaeum+Pyrococcus+horikoshii+OT3+endowed+Escherichia+coli+with+organic+solvent+tolerance.">Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance.</a> Appl. Microbiol. Biotechnol. 79 (3), 443-449 (2008).</li><li>Rehm, H. J., Reiff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+occurrence+of+microbial+oxidation+of+long-chain+alkanes.">Mechanisms and occurrence of microbial oxidation of long-chain alkanes.</a> Adv. Biochem. Eng. / Biotechnol. 19, 175-215 (1981).</li><li>Rojo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+alkanes+by+bacteria.">Degradation of alkanes by bacteria.</a> Environ. Microbiol. 11 (10), 2477-2490 (2009).</li><li>Van Beilen, J. B., Panke, S., Lucchini, S., Franchini, A. G., Rothlisberger, M., Witholt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Pseudomonas+putida+alkane-degradation+gene+clusters+and+flanking+insertion+sequences:+evolution+and+regulation+of+the+alk+genes.">Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.</a> Microbiology. 147, 1621-1630 (2001).</li><li>Wang, L., Tang, Y., Wang, S., Liu, R. L., Liu, M. Z., Zhang, Y., Liang, F. L., Feng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+a+novel+thermophilic+Bacillus+strain+degrading+long-chain+n-alkanes.">Isolation and characterization of a novel thermophilic Bacillus strain degrading long-chain n-alkanes.</a> Extremophiles. 10 (4), 347-356 (2006).</li></ol>

特別な利害関係は宣言されません。

BioBrick原理は、アルカンの分解のために、シャーシを構築するために使われ、このツールキットの単一コンポーネントの原理の証明を得た。いくつかのアッセイは、in vivoおよびアルカン分解する経路酵素のin vitro活性を測定するために提案されている。提示された仕事は、正常宿主生物大腸菌の酵素活性と発現を決定するために使用できるメソッドの数を示しています適?...

Oil pollution is among the most serious causes of environmental contamination, and greatly affects ecosystems, businesses and communities 3. Solutions are for example required to battle the continuous oil pollution originating from the oil sands tailing waters in Alberta, Canada. During the process of oil extraction from oil sands, bitumen, a semi-solid oxidized form of oil, is removed using thermal recovery techniques that consume about 3.1 barrels of water per single barrel of oil 1. Oil contaminated process water, mainly originating from a local river, is stored in tailing ponds after bitumen extraction. A more effective recycling of process water in order to reduce the need for freshwater uptake is needed. To facilitate the bitumen extraction and to ensure that downstream sites meet water quality guidelines for the protection of aquatic ecosystems, process water treatments are rapidly evolving 3.
To treat pollution of organic compounds, bioremediation technologies employing microorganisms are presently encouraged 1. Alkanes are the most abundant family of hydrocarbons in crude oil, containing 5 to 40 carbon atoms per molecule 7, 21. Many bacteria are known to degrade alkanes of various lengths via sequential oxidation of the terminal methyl group forming first alcohols, then aldehydes and finally fatty acids 8. Within this iGEM project several enzymes from different organisms were expressed and characterized, and made available via the BioBrick standard and Registry of Standard Biological Parts.
The well-studied alkane hydroxylase system of Gordonia sp. TF6 facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes using a minimum of four components: alkB2 (alkane 1-monooxygenase), rubA3, rubA4 (two rubredoxins) and RubB (rubredoxin reductase) 8, 21. Oxidation of long-chain alkanes (ranging from C15 up to C36) is reported to be performed by ladA, a flavoprotein alkane monooxygenase from Geobacillus thermodinitrificans NG-80-2 7, 15, 18, 22. LadA forms a catalytic complex with flavin mononucleotide (FMN) that utilizes atomic oxygen for oxidation. This results in the conversion of alkanes into the corresponding primary alkanol. The alcohols are further oxidized by alcohol and aldehyde dehydrogenases to fatty acids, which readily enter the &beta;-oxidation pathway 7, 21. A zinc-independent alcohol dehydrogenase from the thermophillic bacterium Geobacillus thermoleovorans B23 oxidizes medium-chain alkanols into their respective alkanals, using NAD+ as a cofactor 10. Aldehyde dehydrogenase from the same bacterium is able to catalyze the NAD+-dependent final step in the medium-chain oxidation 11.
In order to reduce induction costs and to maintain optimal proliferation of the bacterial system, the promoter pCaiF from E.coli was characterized. This promoter can regulate expression of the hydrocarbon degradation pathway components, and is regulated by cAMP-Crp levels, which in turn depend on glucose levels 6. At high extracellular glucose concentrations in the environment the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) level was low through the inhibition of adenylyl cyclase as a side effect of PTS mediated glucose transport. Conversely, during limitation (low glucose concentrations) the cAMP level increased and Crp bound to cAMP forming the complex, cAMP-Crp, which bound pCaiF and activated transcription of the downstream components 6, 14.
Wildtype E. coli can only tolerate moderate concentrations of hydrocarbons. To complete the toolkit, tolerance to hydrocarbons had to be addressed. Several organic solvent-tolerant bacteria are known to survive in water-solvent two-phase systems 12. Molecular components known to increase tolerance are chaperones that facilitate the correct folding of proteins. The prefoldin system from Pyrococcus horikoshii OT3, consisting of the proteins phPFD&alpha; and phPFD&beta;, was shown to increase hydrocarbon-tolerance 17.
The alkane conversion toolkit was constructed following the BioBrick principle, which is documented at the Registry of Standard Biological Parts 9. BioBricks are plasmids containing a specific functional insert that is flanked by 4 predefined restriction sites. The BioBrick inserts can be extended flexibly, allowing the construction of biological systems with new functions.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>E. coli K12</td>
			<td>New England Biolabs</td>
			<td>C2523H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Octane</td>
			<td>Fluka</td>
			<td>74822</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexadecane</td>
			<td>Fluka</td>
			<td>52209</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>octanol-1</td>
			<td>Fluka</td>
			<td>95446</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dodecanol-1</td>
			<td>Sigma-Aldrich</td>
			<td>126799</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Sigma</td>
			<td>N4505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FMN</td>
			<td>Sigma</td>
			<td>F2253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>J.T. Baker Casno</td>
			<td>7487 889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell disrupter</td>
			<td>LA Biosystems</td>
			<td>CD-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Amersham pharmacia</td>
			<td>spec 2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Tecan</td>
			<td>Magellan v7.0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Innova, 44</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BioBrick K398014: 
			BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4- BBa_J61100-rubB</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a></td>
			<td>Alkane Hydroxylase System 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398027: BBa_R0040-BBa_B0034-ladA</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398027" target="_blank">BBa_K398027</a></td>
			<td>ladA Protein Generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398018: BBa_J23100-BBa_J61101-ADH</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a></td>
			<td>ADH generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398030: BBa_R0040-BBa_B0034-ALDH</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a></td>
			<td>ALDH generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398326: pCaiF</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326</a></td>
			<td>pCaiF promoter 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398331: pCaiF-BBa_B0032-BBa_I13401</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a></td>
			<td>pCaiF measurement device 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398406: BBa_J23002-BBa_J61107-phPFD&alpha;-BBa_J61107-</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a></td>
			<td>Solvent tolerance cluster 
			Resistance: Chloramphenicol</td>
		</tr>
	</tbody>
</table>

a toolkit to enable hydrocarbon conversion in aqueous environments

この作品は、前方の大腸菌によるアルカンの変換を可能にし、その適用性の原理の証明を提示するツールキットを置きます。このツールキットには、複数の標準交換部品（BioBricks）9アルカンの変換、有毒な炭化水素に富む環境における遺伝子発現と生存の調節をアドレッシングで構成されています。 アルカン分解の3段階経路を大腸菌で実装されました大腸菌それぞれアルカノール、カナール、最終的にアルカン-アミノ酸に、中長期鎖アルカンの変換を有効にする。後者はネイティブのβ-酸化経路を介して代謝された。中鎖アルカン（C5〜C13）とシクロアルカン（C5〜C8）、Gordonia spからアルカンヒドロキシラーゼシステムの4つの遺伝子（alkB2、rubA3、rubA4とrubB）の酸化を促進する。 TF6 8,21を 大腸菌に形質転換した大腸菌 。の変換のための長鎖アルカン（C15-C36）は、Geobacillus thermodenitrificansからLADA遺伝子が実装されました。分解プロセスの必要なさらなる手順については、ADHとALDHは（G. thermodenitrificans由来する）10,11を導入しました。活動は休止細胞アッセイにより測定した。各酸化ステップでは、酵素活性が観察された。 プロセス効率を最適化するために、式は唯一、低グルコース条件下で誘導されました：基板-調節プロモーター、pCaiF、使用されていました。 pCaiFは大腸菌に存在している大腸菌 K12と非グルコース炭素源の分解に関与する遺伝子の発現を調節する。 ツールキットの最後の部分-生存を標的に- PhPFDαとβ溶媒耐性遺伝子、 パイロコッカスホリコシ OT3からの両方を使用して実装されていました。有機溶媒としては、否定的にaffectinによって細胞ストレスを誘導し、生存性を低下させる可能性がGタンパク質の折り畳み。シャペロンとして、PhPFDαβはアルカンの存在下でタンパク質の折りたたみのプロセスなどを改善します 。培地で10％n-ヘキサンのプレゼンスの増加成長率（最大50％）で示される炭化水素の改善·トレランスにつながったこれらの遺伝子の発現が観察された。 要約すると、結果は、ツールキットは大腸菌を可能にすることを示す大腸菌水性環境中の炭化水素に変換して耐える。このように、それは合成生物学のアプローチを用いてオイル浄化のための持続的な解決に向けた第一歩を表しています。

Alkane conversion
The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase. 
For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid <a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_...

Watch this Scientific Journal Video about A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments at JoVE.com

水環境での炭化水素変換を可能にするツールキット |バイオエンジニアリング |ビデオ

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

油汚染の浄化のための持続可能な自動調整システムは、標準的な細菌DNAの交換部品（BioBricks）を使用して設計されました。エンジニアリング<em&gt; E大腸菌の</em&gt;株が有毒、水性環境中のβ-酸化を介しアルカンを分解するために使用されていました。異なる種からそれぞれの酵素は、アルカン分解活性を示した。また、に耐性の増加<em&gt; N</em&gt;-ヘキサンをアルカントレラント細菌由来の遺伝子を導入することによって達成された。

水性環境で炭化水素の変換を有効にするツールキット

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

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Alkanes and Cycloalkanes

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14.0K ビュー.  Delft University of Technology. 油汚染の浄化のための持続可能な自動調整システムは、標準的な細菌DNAの交換部品（BioBricks）を使用して設計されました。エンジニアリング E大腸菌の株が有毒、水性環境中のβ-酸化を介しアルカンを分解するために使用されていました。異なる種からそれぞれの酵素は、アルカン分解活性を示した。また、に耐性の増加 N-ヘキサン...

ビデオ: A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

水性二相系を用いた細胞の共培養パターニング

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

TiOを使用して水性環境中のタンパク質や細胞を光パターニング<sub&gt; 2</sub&gt;光触媒

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

マイクロリアクター及び、電磁作動に水滴が酵素として使用

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

三次元培養細胞遊走を特徴付ける傷の環境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

熱と圧力を用いた水性溶媒和による組み換えクモ絹の材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...