この作品は、エネルギー五大湖バイオエネルギー研究センター（科学BER DE - FC02 - 07ER64494のDOE事務所）と米国エネルギー省、基礎エネルギー科学のオフィスからの助成金DE - FG02 - 91ER200021、部門の米国エネルギー省の一部で賄われていた化学科学、地球科学と生命科学。我々は彼らの材料と概念的な貢献のためにジョンスコットクレイグとメリッサBorruschに感謝。

1。酵素の生産<ol><li>ベクトルpPICZまたはpPICZαに対応する遺伝子のクローニングとP.への変換によるピキアパストリス中のタンパク質を生成するピキアパストリス 。メタノール誘導でバッフル付500mlのフラスコに300mlのバッチで細胞を成長させる毎に24時間（バネルジーら、2010A）。 </li><li>タンジェンシャルフロー濾過により培養濾液を濃縮し、脱塩。 </li><li> -80 20％グリセロールで小分けに酵素℃にて保存してください。濃度範囲は1〜10 mg / mlのです。 </li></ol> 2。実験の設計<ol><li>入力テストする酵素の数は、その上部と下部の比率（コア酵素は5％の低い割合など）、およびデザインExpertsoftwareに実験的なデザイン（例えば、二次または三次）のタイプ。 </li><li>の15 mg / gのグルカンの合計負荷を作るに追加する各酵素のμg単位の量を計算し、に追加するμlで各酵素の量を計算するそれぞれの反応。 </li><li>実験的なデザインと分注FXPのソフトウェアにインポートして転送元ファイルの関数を使用してに応じて水と酵素の分注のためにソースの実験器具、ソースの井戸、先の実験器具、先の井戸、および移動量を指定してExcelワークシートを生成します。 </li></ol> 3。酵素加水分解<ol><li>パドルの貯水池で、5μg/ mlのテトラサイクリン、5μg/ mlのシクロヘキシミドを含む、50mMのクエン酸ナトリウム、pHを4.8にバイオマスのスラリーを一時停止します。 </li><li>スパン- 8カットオフ最後の0.5センチメートル1000 -μLピペットチップを用いて96ウェルディープウェル反応プレートの各ウェルにスラリーのロボット制御でピペットを200μl。を100μl/秒に一度スラリーを混合してから、パドルの貯水池から同じボリュームを吸引し、プレートに分注する。 </li><li>ベックマンFXに入力されたプログラムを使用して、同じプレートに酵素を分注する。 </li><li>貫​​通capmatsでプレートをシール。 </li><li>逆さにしてタッププレート表面張力を克服するために数回。 10rpmでハイブリダイゼーションインキュベーター回転で48時間、50℃でプレートを置きます。 </li></ol> 4。グルコースとXYLの決定<ol><li>スイングバケット遠心機で1250 × gで3分間反応プレートを遠心する。 </li><li>分注FXのAP96ポッドを使用して、通常の96ウェルプレートに上清100μlを移す。 </li><li> 90から95でプレートをインキュベート℃で10分間の酵素を不活性化し、30秒、1250 × gで遠心分離するために。 </li><li>グルコースの測定では、定期的な96ウェルプレートに上清12μlを移す。グルコースオキシダーゼ/ペルオキシダーゼ（GOPOD）試薬192μlを添加する。 20分間50℃で培養すると、マイクロプレートリーダーで510 nmで吸光度を読み取る。ブランクは、酵素なしで反応混合物です。 </li><li> XYL測定では、384ウェルプレートに4μlを移す。アッセイ試薬を追加し、340nmでプレートを読み取る。空酵素なしで反応混合物です。 </li></ol> 5。データ解析<ol><li>既知のグルコースと、元のバイオマスのXYL内容に基づいて％グルコースとXYLの収量に吸光度の値を変換します。デザインエキスパートソフトウェアへの応答として、これらのパーセンテージをインポートします。堅牢なモデルのための基準が満たされていることを確認するために統計的paramentersを確認してください。 </li><li>実験的に最適なモデルの予測を確認してください。 </li></ol> 6。 GENPLATと代表的な結果： （1）個々の純粋なタンパク質の最適化された混合物の作成 ​​。結果は、酵素が重要であるかを示し、そしてどのような割合で、そしてまたその酵素は重要ではありません 。 T.に豊富に存在するいくつかのタンパク質このようなCIP1とCIP2としてトリコデルマリーセイ secretome（Nagendranら、2009）、、アンモニア繊維の拡大（AFEX）またはアルカリ俵で前処理したトウモロコシの茎葉からグルコースのリリースでは何の役割も果たさないように見える<html>ロゲンの過酸化水素（AHP）（バネルジーら、2010B）。一方、いくつかのタンパク質が突然重要です。例えば、糖加水分解酵素ファミリー10と11のエンド - キシラナーゼはGlcのリリースの両方が重要であり、一つキシラナーゼは、他の（。バネルジーら、2010B、C）のために代用することはできません。 Cel61A、T.のマイナータンパク質XYLのリリースでは、 トリコデルマリーセイ secretomeを、グルコースは非常に重要ですが、ではない。他の人がどちらか一方だけ（バネルジーら、2010C）のために必要であるのに対し、いくつかの酵素は、グルコースとXYLの両方のリリースのために重要である。 16成分、0.94 mg / mlの（すなわち、最適化されていない混合物）の濃度で各々を含有する合成混合物は、AFEX前処理DDGから利用可能なグルコースの38％を発表した。同一の加水分解条件下では、最適化された混合物は、これで16成分の濃度は、0％から32％であった（図2）グルコースの52％を発表した。この実験では、定義された混合物を構築するユーティリティを示しています。 &quot;万能サイズ&quot;&gt;（2）別の前処理/基板の組み合わせに最適化されたカクテルの作成 ​​。現在の商業セルラーゼの準備がある&quot;_content。現実には、最高のカクテルは、基板と前処理の両方に依存する。単一製品Accellerase 1000と同じ方法で前処理した別の基板から別の収量を与え、別の前処理（図1）に公開されている同じ基板からそのように我々は、複数の前処理とみとめ原料（バネルジーらのために純粋な酵素の混合物を最適化するためにGENPLAT使用しているら、2010C）。図2は、16純粋な酵素の最適な酵素比率がAFEX前処理トウモロコシの茎葉、AHP -前処理トウモロコシの茎葉、およびAFEX前処理DDGの違いを示しています。わずか11酵素を図2に示されているの最適な理由他の五（Cel61B、AbfB、CIP1、CIP2、およびCel12A）の割合は0％（バネルジーら、2010C）であることがわかった。 （3）新規酵素の発見 </strオング&gt;。純粋なタンパク質の最適化された混合物の建設は継続的なプロセスです。新しいタンパク質は（商業的供給源から、異種発現によって、または精製して）純粋な形で利用できるようになると、それらは体系的に現在最適化されたセットに追加してテストすることができます。グルコースまたはXYLに寄与する新規タンパク質は、新しい、最適化されたセットの一部となってから離します。このように、GENPLATはバイオマスの脱構築のための新しい酵素を同定するためのプラットフォームになります。我々は、T.以外の菌の抽出を行っているデルマリーセイおよびA.ニジェールでは、異なる基板上に成長した。私たちのコアセットに追加したとき、ある特定の抽出は、グルコース収率を後押しした。責任あるタンパク質を精製し、（Banerjeeさんとウォルトン、未発表結果）任意の市販の酵素調製物中に存在する新規なグリコシル加水分解酵素であることが示された。 よりよい酵素の（4）発見 。の劣化のための酵素が最も重要であるGENPLATで表示した特にバイオマスは、私たちは酵素が改善のための焦点でなければならないそのうちのよりよい理解を持っている。重要な酵素の良い例は、自然の中で検索して見つけるか、タンパク質工学によって作ることができる。 （酵素は、所望の特性に応じて、いくつかの方法でそのT.デルマリーセイのホモログ、より&quot;良い&quot;かもしれない、例えば、より高い比活性、より大きな相乗効果は、タンパク質分解感受性、または強化された熱安定性を減少させた）。純粋なセルロースまたは他の人工的な基質との結果が自然リグノセルロースに該当しない場合がありますので、それは現実的に複雑なカクテル（重要ではバイオマスの酵素は、単独で動作しないため）と現実的な基板（重要を使用しているためGENPLATは、潜在的に優れた酵素をアッセイするために意味のあるプラットフォームを提供します。材料）。 （5）市販の酵素の最適化 。純粋な酵素の十分に大きな量はまだ現実の世界には存在しません。彼らが実行するまで、エタノールプロducersなどAccellerase 1000 MultifectXylanaseとして商用製品に依存する必要があります。しかし、市販酵素の混合物は一般に、個々のものよりパフォーマンスに優れ、そしてGENPLATは、複数の市販酵素の最適化されたカクテルを設計するために使用することができます。図3は、4つの市販の酵素製剤の混合物を最適化するためにGENPLATの使用方法を示します。 AFEX前処理DDGからGlcのリリースのための最適な割合は47％Accellerase1000、27％Multifectペクチナーゼ、22％Novozyme 188、および4％Multifectキシラナーゼ（バネルジーら、2010C）であることがわかった。図3Bは、4つの酵素製剤とAFEX - DDGからグルコース収率の違いを示しています。最適化された商業用混合物は、高グルコースの収率が得られるだけでAccellerase1000より、SpezymeCP単独で、または16ビットコンポーネントの合成混合物。この実験はまた、16成分合成混合物は、市販酵素の一つ以上に存在する最適なグルコース放出、のために必要な1つ以上の酵素が欠けていることを示しています。 GENPLATは、私たちすることができますそのようなコンポーネントを識別するためにED。 <img src="/files/ftp_upload/3314/3314fig1.jpg" alt="図1"/> 図1は、単一の市販の酵素調製物は、すべての基板と、すべての前処理に最適なです 。五基板三前処理は、（0.5 mmの粒径）研削の同様の条件下で比較した、酵素負荷（15 mg / gのグルカン）、および加水分解の条件（48時間、50 ° C）異なるAFEX前処理基板の。A.消化 Accellerase 1000で。トウモロコシの茎葉のB.の消化Accellerase 1000で3つの異なる方法（Banerjeeさんらを参照してください。、2010C）で前処理する。 <img src="/files/ftp_upload/3314/3314fig2.jpg" alt="図2"/> 図2。AFEX前処理トウモロコシの茎葉（AFEX - CS）、アルカリ性過酸化水素前処理トウモロコシの茎葉（AHP - CS）、またはAFEX前処理乾燥は蒸留からのグルコースの放出のための11の酵素の最適な比率ERS&quot;穀物（AFEX - DDG）。データは、バネルジーらからです。 （2010C）。バーの上部にある数字は、バイオマス試料中の全Glcのパーセントとしてグルコースの収量です。 <img src="/files/ftp_upload/3314/3314fig3.jpg" alt="図3"/> 図3。A. AFEX前処理DDGからGlcのリリースの4つの市販の酵素製剤の最適化された混合物を生成するためにGENPLATの使用。 Multifectキシラナーゼは、最小の貢献（4％）したため、この三角図から省略した。B.グルコースの収率は、同一の加水分解条件下での異なる酵素処理（15 mg / gのグルカンの酵素、48時間、50℃）でAFEX - DDGから。 &quot;四成分の商業は、&quot;図に示すように実験から決定されたプロポーションを持っています。 3A。

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利害の衝突は宣言されません。

これは、広く酵素のコストを削減することが経済的なリグノセルロースエタノール産業の発展にとって重要であることが認識されている。現在入手可能な市販の酵素カクテルは、多くのタンパク質（Nagendranら、2009）の複雑で不完全に定義された混合物であり、これらは主に酸で前処理トウモロコシの茎葉上での使用に適合している。優れた酵素カクテルの開発を加速するためには、いくつかの研究室では、酵素の発見と特性評価のためのハイスループットプラットフォームを開発しました。 。。デッカーらロボットの酵素とバイオマススラリーの調剤、実験の統計的設計、および/またはグルコースの自動測定とXYL（ベルリンら、2007：この地域での取り組みもGENPLATで見つかった次のプロパティの一つ以上を組み込んでいますら、2009;。Kimら、1998;。キングら、2009）。。 GENPLATはに最大で6つのコンポーネントから分析することができる酵素の混合物の複雑さで最も大きく、これらの以前の取り組みを拡張する私たちの最新作で16個以上の初期の研究（バネルジーら、2010C）。穏やかに転倒回転による消化中に混合;とGluとXYLの自動比色定量GENPLATのその他の主要機能は、調剤中に中断された茎葉のスラリーを保つことができるビーズ混合室の使用（パドルの貯水池）です。

使用機材とソフトウェア： <ol><li>デザインエキスパートソフトウェア、バージョン8.0（スタット使いやすさ、（株）、ミネアポリス、ミネソタ州）（ <a href="http://www.statease.com">http://www.statease.com</a> ） </li><li>分注FXPラボ自動化ワークステーション（Beckman - Coulter社、フラートン、カリフォルニア州） </li><li>分注FXPソフトウェア、バージョン3.3（Beckman - Coulter社、フラートン、カリフォルニア州） </li><li>パドルの貯水池（モデルVP 756C - 1P100、V＆Pサイエンティフィック社、サンディエゴ） </li><li>ウェルプレート、96ディープ（1.5 ml）を（Abgene、フィッシャーサイエンティフィック、エプソン、英国） </li><li>貫​​通capmats（米国サイエンティフィック、オカラ、フロリダ州） </li><li>回転ハイブリダイゼーションインキュベーター（モデル5420、VWR） </li><li>スイングローター遠心機（モデル5810R、エッペンドルフ、ハンブルク、ドイツ） </li><li>プレート、コスター96ウェル（Abgene、フィッシャーサイエンティフィック、エプソン、英国） </li><li> 384ウェルプレート、（BDファルコン、BD Biosciences社、サンノゼ、カリフォルニア州） </li></ol>使用される試薬： <ol><li> ピキアパストリスとベクトル：ひずみX33、およびpPICZとpPICZα（Invitrogen社、カールスバッド、カリフォルニア州） </li><li> T.デルマリーセイとベクトル：ひずみQM9414（農業利用研究のための農業ナショナルセンター、ピオリア、イリノイ州の米国エネルギー省）、およびpCB1004 - EV（Banerjeeさんら、2010B。） </li><li> Accellerase 1000（ロット番号1600844643）（ジェネンコア社、ロチェスター、ニューヨーク州） </li><li> 50mMのクエン酸ナトリウム緩衝液、pH 4.8 </li><li>テトラサイクリン在庫：10 mg / mlの</li><li>シクロヘキシミドの株式：10 mg / mlの</li><li> GOPODアッセイキット、カタログK - GLUC（Megazyme、ブレイ、アイルランド） </li><li>キシロースアッセイキット、カタログK -キシロース（Megazyme、ブレイ、アイルランド） </li></ol>

genplat: an automated platform for biomass enzyme discovery and cocktail optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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Watch this Scientific Journal Video about GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization at JoVE.com

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT（GLBRC酵素のプラットフォーム）は、バイオマスの分解のための酵素カクテルの発見と最適化のための自動化されたプラットフォームです。それは、複数のコンポーネントが含まれている酵素の複数の原料と混合物に適合させることができます。

GENPLAT：バイオマスの酵素の発見とカクテルの最適化のための自動化プラットフォーム

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

genplat-an-automated-platform-for-biomass-enzyme-discovery-cocktail

Research

JoVE Journal

Bioengineering

15.3K ビュー.  Michigan State University. GENPLAT（GLBRC酵素のプラットフォーム）は、バイオマスの分解のための酵素カクテルの発見と最適化のための自動化されたプラットフォームです。それは、複数のコンポーネントが含まれている酵素の複数の原料と混合物に適合させることができます。

ビデオ: GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

バイオマテリアルのハイスループット検出用高分子マイクロアレイ

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

生物工学

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

酵素の固定化と安定化のための疎水性の塩で修飾ナフィオン

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

マイクロスケール細胞刺激のための実験のための多目的自動化プラットフォーム

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

開発<em&gt;インビトロ</em&gt;眼プラットフォームはコンタクトレンズをテストします

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

微生物光遺伝学アプリケーションのための自動照明、培養、およびサンプリングシステムの設計と実装

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

パフォーマンス テスト用 FR 4 銅クラッド電極に伝導マイクロ ポンプ

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

自動 Microbioreactor 技術を用いた異種タンパク質生産の最適化のための汎用プロトコル

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

ラットでの肝部分工学生体内での基盤として新しい手術手技

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

化学のエピジェネティックな修飾剤の細胞機械装置をリダイレクトすることによって遺伝子の転写を抑制

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...