私たちは便利な議論や編集のためのプロトコル、キーラン・スウィーニーをテストする際に支援するためのモリーラザルとベロニカ・デルガドに感謝して、原稿の重要な読書のためのテイラー・スコット、私のアン・adirekkun、およびステファニーゲラーだろう。ミーガンニコールMcClean氏、博士バローズウェルカム基金からの科学的なインターフェイスでのキャリア賞を保持しています。

<ol>
	<li>Motta-Mena, L. B., Reade, A., 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=An+optogenetic+gene+expression+system+with+rapid+activation+and+deactivation+kinetics.">An optogenetic gene expression system with rapid activation and deactivation kinetics.</a> Nature chemical biology. 10 (3), 196-202 (2014).</li><li>Hughes, R. M., Bolger, S., Tapadia, H., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-mediated+control+of+DNA+transcription+in+yeast.">Light-mediated control of DNA transcription in yeast.</a> Methods. 58 (4), 385-391 (2012).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature biotechnology. 20 (10), 1041-1044 (2002).</li><li>Polstein, L. R., Gersbach, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Light-inducible+spatiotemporal+control+of+gene+activation+by+customizable+zinc+finger+transcription+factors.">a Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors.</a> J Am Chem Soc. 134 (40), 16480-16483 (2012).</li><li>Polstein, L. R., Gersbach, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+A+light-inducible+CRISPR-Cas9+system+for+control+of+endogenous+gene+activation.">a A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.</a> Nature Chemical Biology. 11 (3), 198-200 (2015).</li><li>Yang, X., Jost, A. P. T., Weiner, O. D., Tang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-inducible+organelle-targeting+system+for+dynamically+activating+and+inactivating+signaling+in+budding+yeast.">A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast.</a> Molecular biology of the cell. 24 (15), 2419-2430 (2013).</li><li>Lee, S., Park, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+protein+inactivation+by+optogenetic+trapping+in+cells.">Reversible protein inactivation by optogenetic trapping in cells.</a> Nature methods. 11 (6), 633-636 (2014).</li><li>Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nature Methods. 7 (12), 973-975 (2010).</li><li>Yazawa, M., Sadaghiani, A. M., Hsueh, B., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+protein-protein+interactions+in+live+cells+using+light.">Induction of protein-protein interactions in live cells using light.</a> Nat Biotechnol. 27 (10), 941-945 (2009).</li><li>Zoltowski, B. D., Motta-Mena, L. B., Gardner, 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=Blue+light-induced+dimerization+of+a+bacterial+LOV-HTH+DNA-binding+protein.">Blue light-induced dimerization of a bacterial LOV-HTH DNA-binding protein.</a> Biochemistry. 52 (38), 6653-6661 (2013).</li><li>Renicke, C., Schuster, D., Usherenko, S., Essen, L. O., Taxis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+LOV2+Domain-Based+Optogenetic+Tool+to+Control+Protein+Degradation+and+Cellular+Function.">A LOV2 Domain-Based Optogenetic Tool to Control Protein Degradation and Cellular Function.</a> Chemistry &amp; Biology. 20 (4), 619-626 (2013).</li><li>Novick, A., Szilard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+of+the+chemostat.">Description of the chemostat.</a> Science (New York, N.Y.). 112 (2920), 715-716 (1950).</li><li>Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=La+technique+de+culture+continue.+Th&#233;orie+et+applications.">La technique de culture continue. Th&#233;orie et applications.</a> Ann. Inst. Pasteur. 79 (4), 390-410 (1950).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature Biotechnology. 20, 1041-1044 (2002).</li><li>Kennedy, M. J., Hughes, R. M., Peteya, L. A., Schwartz, J. W., Ehlers, M. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nature Methods. 7, 973-975 (2010).</li><li>Olson, E. J., Tabor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+characterization+methods+overcome+key+challenges+in+synthetic+and+systems+biology.">Optogenetic characterization methods overcome key challenges in synthetic and systems biology.</a> Nature chemical biology. 10 (7), 502-511 (2014).</li><li>Botstein, D., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast:+An+experimental+organism+for+21st+century+biology.">Yeast: An experimental organism for 21st century biology.</a> Genetics. 189 (3), 695-704 (2011).</li><li>Galanie, S., Smolke, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+yeast-based+production+of+medicinal+protoberberine+alkaloids.">Optimization of yeast-based production of medicinal protoberberine alkaloids.</a> Microbial cell factories. 14 (1), 144 (2015).</li><li>Gerngross, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+production+of+human+therapeutic+proteins+in+yeasts+and+filamentous+fungi.">Advances in the production of human therapeutic proteins in yeasts and filamentous fungi.</a> Nature biotechnology. 22 (11), 1409-1414 (2004).</li><li>van Maris, A. J. A., Abbott, D. A., 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=Alcoholic+fermentation+of+carbon+sources+in+biomass+hydrolysates+by+Saccharomyces+cerevisiae:+current+status.">Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status.</a> Antonie van Leeuwenhoek. 90 (4), 391-418 (2006).</li><li>Edelstein, A., Amodaj, N., Hoover, K., Vale, R., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+control+of+microscopes+using+&#181;Manager.">Computer control of microscopes using &#181;Manager.</a> Curr Protoc Mol Biol. , (2010).</li><li>Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+of+microscope+control+using+&#181;Manager+software.">Advanced methods of microscope control using &#181;Manager software.</a> Journal of biological methods. 1 (2), (2014).</li><li>Schindelin, J., Arganda-Carreras, I., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature methods. 9 (7), 676-682 (2012).</li><li>Bhattacharya, S., Datta, A., Berg, J. M., Gangopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+surface+wettability+of+poly(dimethyl)+siloxane+(PDMS)+and+glass+under+oxygen-plasma+treatment+and+correlation+with+bond+strength.">Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength.</a> Journal of Microelectromechanical Systems. 14 (3), 590-597 (2005).</li><li>Saldanha, A. J., Brauer, M. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+homeostasis+in+batch+and+steady-state+culture+of+yeast.">Nutritional homeostasis in batch and steady-state culture of yeast.</a> Molecular Biology of the Cell. 15, 4089-4104 (2004).</li><li>Boer, V. M., Tai, S. L., 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=Transcriptional+responses+of+Saccharomyces+cerevisaie+to+preferred+and+nonpreferred+nitrogen+sources+in+glucose-limited+chemostat+cultures.">Transcriptional responses of Saccharomyces cerevisaie to preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures.</a> FEMS Yeast Research. 7, 604-620 (2007).</li><li>Boer, V. M., Amini, S., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+genotype+and+nutrition+on+survival+and+metabolism+of+starving+yeast.">Influence of genotype and nutrition on survival and metabolism of starving yeast.</a> Proceedings of the National Academy of Sciences. 105, 6930-6935 (2008).</li><li>Brauer, M. J., Huttenhower, C., 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=Coordination+of+growth+rate,+cell+cycle,+stress+response+and+metabolic+activity+in+yeast.">Coordination of growth rate, cell cycle, stress response and metabolic activity in yeast.</a> Molecular Biology of the Cell. 19, 352-367 (2008).</li><li>Gresham, D., Dunham, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enduring+utility+of+continuous+culturing+in+experimental+evolution.">The enduring utility of continuous culturing in experimental evolution.</a> Genomics. 104 (6), 399-405 (2014).</li><li>Liu, H., Yu, 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=Photoexcited+CRY2+interacts+with+CIB1+to+regulate+transcription+and+floral+initiation+in+Arabidopsis.">Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.</a> Science (New York, N.Y.). 322 (5907), 1535-1539 (2008).</li><li>McIsaac, R. S., Oakes, B. L., Wang, X., Dummit, K. A., Botstein, D., Noyes, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+gene+expression+perturbation+systems+with+rapid,+tunable,+single-gene+specificity+in+yeast.">Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.</a> Nucleic Acids Research. 41 (4), 1-10 (2013).</li><li>McIsaac, R. S., Petti, A. A., Bussemaker, H. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation-based+analysis+and+modeling+of+combinatorial+regulation+in+the+yeast+sulfur+assimilation+pathway.">Perturbation-based analysis and modeling of combinatorial regulation in the yeast sulfur assimilation pathway.</a> Molecular biology of the cell. 23 (15), 2993-3007 (2012).</li><li>McIsaac, R. S., Silverman, S. J., 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=Fast-acting+and+nearly+gratuitous+induction+of+gene+expression+and+protein+depletion+in+Saccharomyces+cerevisiae.">Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.</a> Molecular biology of the cell. 22 (22), 4447-4459 (2011).</li><li>Melendez, J., Patel, M., Oakes, B. L., Xu, P., Morton, P., McClean, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optogenetic+control+of+intracellular+protein+concentration+in+microbial+cell+cultures.">Real-time optogenetic control of intracellular protein concentration in microbial cell cultures.</a> Integrative biology quantitative biosciences from nano to macro. 6 (3), 366-372 (2014).</li><li>Brauer, M. J., Huttenhower, C., 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=Coordination+of+growth+rate,+cell+cycle,+stress+response,+and+metabolic+activity+in+yeast.">Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast.</a> Molecular biology of the cell. 19 (1), 352-367 (2008).</li><li>Boer, V. M., Crutchfield, C. A., Bradley, P. H., Botstein, D., Rabinowitz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-limiting+intracellular+metabolites+in+yeast+growing+under+diverse+nutrient+limitations.">Growth-limiting intracellular metabolites in yeast growing under diverse nutrient limitations.</a> Molecular biology of the cell. 21 (1), 198-211 (2010).</li><li>Markham, B. E., Byrne, W. J., Methods, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+storage+and+utilization+of+phosphate+by+yeast.">Uptake, storage and utilization of phosphate by yeast.</a> Journal of the Institute of Brewing. 73, 271-273 (1967).</li><li>Kazemi Seresht, A., Palmqvist, E. A., Olsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+phosphate+scarcity+on+pharmaceutical+protein+production+in+S.+cerevisiae:+linking+transcriptomic+insights+to+phenotypic+responses.">The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses.</a> Microbial cell factories. 10 (1), 104 (2011).</li><li>Shimizu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+Systems+of+Bacteria+such+as+Escherichia+coli+in+Response+to+Nutrient+Limitation+and+Environmental+Stresses.">Regulation Systems of Bacteria such as Escherichia coli in Response to Nutrient Limitation and Environmental Stresses.</a> Metabolites. 4 (1), 1-35 (2013).</li><li>Olson, E. J., Hartsough, L. A., Landry, B. P., Shroff, R., Tabor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+bacterial+gene+circuit+dynamics+with+optically+programmed+gene+expression+signals.">Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals.</a> Nature methods. 11 (4), 449-455 (2014).</li></ol>

著者らは、開示することは何もありません。

私たちは心の中で柔軟にこの装置を設計しました。使用されるすべてのコードはフリーでオープンソースです。セグメント細胞へのデフォルトの画像解析処理が簡単で、すぐに実行されます。カスタム解析は、フィジーグラフィック・ユーザ・インタフェースを代表画像を解析BeanShellのスクリプトに入力を変換し、その後、スクリプトを呼び出すためのプラグインを設定する際に、ユーザ入?...

光遺伝学システムは、遺伝子発現、1、2、3、4、5タンパク質の局在化、6タンパク質活性、6、7、8タンパク質結合、8、9、10、およびタンパク質分解を含む細胞プロセスの成長のリストを制御するために光を使用します。プログラムされた光刺激と制御された環境で細胞を培養するため、および生物学的に関連するタイムスケールの上にそれらの応答を測定するための11の方法は、細胞生物学、バイオテクノロジーの研究のためにこれらのツールの可能性を利用することが必要です。我々の方法は、AE、よく混合した一定の細胞増殖速度を維持するためにchemostasisを利用します評価、及びプログラムされた照明に曝される温度制御されたガラス製の培養容器12、13。倒立顕微鏡で培養排水中の我々の画像の個々の細胞は、プログラムされた照明への文化の応答を測定します。流出液の細胞培養の蛍光強度および細胞形態は、ユーザ入力なしに複数日にわたって測定することができるように培養、サンプリング、画像化、および画像解析は完全に自動化されています。 このプロトコルは、増殖する細胞培養及び顕微鏡精通ほとんどの研究室で実施することができ、使用される装置は、安価で容易に入手可能なコンポーネントで構成されています。透明な培養容器は、光の1μW/ cm 2の範囲では-10 mW / cm 2で発光可能な発光ダイオード（LED）のマトリックス上に配置されています。微生物を連続培養容器で増殖させます。 1蠕動ポンプがでメディアを追加するために使用されます希釈率は、他の顕微鏡に小さい速度で培養物を回収するために使用され、その差は、オーバーフロー出口を通って逃げます。加熱パッドは、温度を維持しています。空気が継続的に正の圧力を維持するだけでなく、ミックスと文化を通気するために培養容器内に圧送されます。空気ポンプを除き、これらのデバイスへの電力はまた、温度計と接続されたデスクトップコンピュータからの入力を受け取るマイクロコントローラによって調節されます。流出液の細胞培養物を倒立顕微鏡のステージ上のマイクロ流体デバイスに圧送されます。非蛍光及び蛍光画像を自動的に取得されます。画像中の細胞は、関心領域（ROI）のように各セルを検索し、各ROIの特性を測定するアルゴリズムを特徴とします。 このプロトコルの適用を実証するために、我々は、青色光responsiで操作サッカロミセス・セレビシエ（Saccharomyces cerevisiae）細胞の変化する光強度に対する応答を測定し蛍光タンパク質の転写を制御する光遺伝学システムまし。このシステムにおいて遺伝子発現を制御するための複数の光遺伝学系はすでに14、15、16が存在するため、一般的パン酵母としても知られているS.セレビシエは 、選択されました。さらに、このモデル生物は、一般にシステム生物学17およびバイオテクノロジーアプリケーション18、19、20のための筐体としての研究のために使用されます。私たちの代表的な結果は、このプロトコルは、入力光強度を変化させると、蛍光レポーターの産生を測定することによって、複数日間の培養の転写を制御するために使用することができることを示しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Extensive lab manual</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >An extensive, regularly updated lab manual is available in the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files). This also includes a description of the microfluidic mold used to generate the representative results.</td>
		</tr>
		<tr >
			<td >Fritzing Design Viewer</td>
			<td >Fritzing</td>
			<td >NA</td>
			<td >The free, open-sourced software to view and edit the .fzz type circuit board designs is available at &#34;http://fritzing.org/download/&#34;</td>
		</tr>
		<tr >
			<td >Arduino Uno R3 (Atmega328 - assembled)</td>
			<td >Adafruit</td>
			<td >50</td>
			<td >Microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >Arduino Stackable Header Kit</td>
			<td >SparkFun Electronics</td>
			<td >10007</td>
			<td >Female pin headers for connecting PCB to microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >Adjustable 30W 110V soldering iron - XY-258 110V</td>
			<td >Adafruit</td>
			<td >180</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >Soldering iron stand</td>
			<td >Adafruit</td>
			<td >150</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >Mini Solder spool - 60/40 lead rosin-core solder 0.031&#34; diameter - 100g</td>
			<td >Adafruit</td>
			<td >145</td>
			<td >For making electrical connections to the PCB. 1 required.</td>
		</tr>
		<tr >
			<td >0.1 &#956;F capacitor</td>
			<td >SparkFun Electronics</td>
			<td >COM-08375</td>
			<td >Stabilizes voltage in PCB. 1 required.</td>
		</tr>
		<tr >
			<td >10 &#956;F capacitor</td>
			<td >SparkFun Electronics</td>
			<td >COM-00523</td>
			<td >Stabilizes voltage in PCB. 1 required.</td>
		</tr>
		<tr >
			<td >MAX7219CNG LED Matrix/Digit Display Driver - MAX7219</td>
			<td >Maxim</td>
			<td >MAX7219CNG</td>
			<td >LED driver. 1 required.</td>
		</tr>
		<tr >
			<td >8 pin IC Socket</td>
			<td >Mouser Electronics</td>
			<td >575-144308</td>
			<td >16 required. These will be stacked on top of each other to support the culture vessel above the LED matrix.</td>
		</tr>
		<tr >
			<td >24 Pin IC socket</td>
			<td >Mouser Electronics</td>
			<td >535-24-3518-10</td>
			<td >Optional. Use this to reversibly attach the MAXIM 7219CNG driver to the PCB.</td>
		</tr>
		<tr >
			<td >Digital multimeter</td>
			<td >Adafruit</td>
			<td >2034</td>
			<td >For troubleshooting electronics. 1 required.</td>
		</tr>
		<tr >
			<td >Break Away Headers - 40-pin Male (Long Centered, PTH, 0.1&#34;)</td>
			<td >SparkFun Electronics</td>
			<td >PRT-12693</td>
			<td >Male pin headers for connected LED matrix to printed circuit board. Ends can be trimmed with wire cutters. 1 set required.&#160;</td>
		</tr>
		<tr >
			<td >Flush diagonal wire cutters</td>
			<td >Adafruit</td>
			<td >152</td>
			<td >For trimming long pin headers and cutting power cables. 1 required.</td>
		</tr>
		<tr >
			<td >Premium Female/Female Jumper Wires - 40 x 12&#34; (300mm)</td>
			<td >Adafruit</td>
			<td >793</td>
			<td >Wire ribbon for connecting breadboard to LED matrix. Can be connected end-to-end with male pin-headers to be longer. 1 required.</td>
		</tr>
		<tr >
			<td >Half-size breadboard</td>
			<td >Adafruit</td>
			<td >64</td>
			<td >The LED matrix will connect to this and the culturing vessel will rest above it.</td>
		</tr>
		<tr >
			<td >Miniature 8x8 Blue LED Matrix</td>
			<td >Adafruit</td>
			<td >956</td>
			<td >Light source. Dominant wavelength is 470nm (blue). 1 required. Alternative miniature LED matrices from the same vendor are available with dominant wavelengths: 624 nm (red), 588 nm (yellow), 525 nm (green), 572 nm (yellow-green), and white.</td>
		</tr>
		<tr >
			<td >Stackable header-3 pin</td>
			<td >SparkFun Electronics</td>
			<td >13875</td>
			<td >8 required.</td>
		</tr>
		<tr >
			<td >Resistor Kit - 1/4W (500 total)</td>
			<td >SparkFun Electronics</td>
			<td >10969</td>
			<td >For electronics. 1 required.</td>
		</tr>
		<tr >
			<td >&#160;IRL520N MOSFET</td>
			<td >International Rectifier</td>
			<td >IRL520N</td>
			<td >Voltage regulating switch for controlling DC current. 4 required.</td>
		</tr>
		<tr >
			<td >Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td>
			<td >SparkFun Electronics</td>
			<td >PRT 11367</td>
			<td >Wire for electronics. 1 required.</td>
		</tr>
		<tr >
			<td >5V 2A (2000mA) switching power supply - UL Listed</td>
			<td >Adafruit</td>
			<td >276</td>
			<td >Power supply for the heating pad and Arduino. 2 required.</td>
		</tr>
		<tr >
			<td >12 VDC 1000mA regulated switching power adapter - UL listed</td>
			<td >Adafruit</td>
			<td >798</td>
			<td >For peristaltic pumps. 2 required.</td>
		</tr>
		<tr >
			<td >Electric Heating Pad - 10cm x 5cm</td>
			<td >Adafruit</td>
			<td >1481</td>
			<td >For heating the bioreactor. 1 required.</td>
		</tr>
		<tr >
			<td >Low flow variable flow peristaltic pump</td>
			<td >Fisher Scientific</td>
			<td >13-876-1</td>
			<td >For pumping media. 1 required</td>
		</tr>
		<tr >
			<td >Medium flow variable flow peristaltic pump</td>
			<td >Fisher Scientific</td>
			<td >13-876-2</td>
			<td >For pumping culture. 1 required.</td>
		</tr>
		<tr >
			<td >9 VDC 1000mA regulated switching power adapter - UL listed</td>
			<td >Adafruit</td>
			<td >63</td>
			<td >For microcontroller power supply. Order 1.</td>
		</tr>
		<tr >
			<td >High Temp Waterproof DS18B20 Digital temperature sensor + extras</td>
			<td >Adafruit</td>
			<td >642</td>
			<td >Thermometer for the bioreactor. 1 required.</td>
		</tr>
		<tr >
			<td >Micromanager</td>
			<td >Micromanager</td>
			<td >NA</td>
			<td >The free, open-sourced microscope control software is available at &#34;https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release&#34;</td>
		</tr>
		<tr >
			<td >FIJI</td>
			<td >ImageJ</td>
			<td >NA</td>
			<td >The free, open-sourced image analysis software is available at &#34;http://fiji.sc/&#34;</td>
		</tr>
		<tr >
			<td >Arduino Integrated Development Environment</td>
			<td >Arduino</td>
			<td >NA</td>
			<td >The free, open-sourced IDE is available at &#34;https://www.arduino.cc/en/Main/Software&#34;</td>
		</tr>
		<tr >
			<td >Custom code</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >The custom microcontroller code and &#34;Bioreactor Controller&#34; plugin are available in the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td>
		</tr>
		<tr >
			<td >USB Cable A to B - 6 Foot</td>
			<td >SparkFun Electronics</td>
			<td >CAB-00512</td>
			<td >Used to download data to microcontroller. 1 required.</td>
		</tr>
		<tr >
			<td >bioreactorTimecourse_example.csv</td>
			<td >GitHub</td>
			<td >NA</td>
			<td >The advantage of loading LED matrix values from a CSV file is that a program can be called by the plugin to update those values based on image analysis results, and those values can be reloaded to the microcontroller, enabling feed-back control. It is available from the &#8220;Optogenetic Chemostat Files&#8221; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td>
		</tr>
		<tr >
			<td >Tota-frost gels (diffusion paper)</td>
			<td >B&#38;H</td>
			<td >B&#38;H # LOFSFTL 
			MFR # T1-72</td>
			<td >For LED matrix. 1 required.</td>
		</tr>
		<tr >
			<td >Kitting Sheet Crosslink 1/4x12x24in</td>
			<td >Grainger, inc</td>
			<td >20JL37</td>
			<td >Black foam for culturing vessel enclosure. 4 required.</td>
		</tr>
		<tr >
			<td >Standard Photodiode Power Sensor, Si, 200 - 1100 nm, 50 mW&#160;</td>
			<td >Thorlabs</td>
			<td >S120VC</td>
			<td >For measuring light intensity. 1 required.</td>
		</tr>
		<tr >
			<td >Labelling Tape</td>
			<td >Fisher Scientific</td>
			<td >159015N</td>
			<td >For labelling and securing loose components. 1 required.</td>
		</tr>
		<tr >
			<td >Compact Power and Energy Meter Console, Digital 4&#34; LCD</td>
			<td >Thorlabs</td>
			<td >PM100D</td>
			<td >For measuring light intensity. 1 required.</td>
		</tr>
		<tr >
			<td >100mL GL45 hybridization glass bottle</td>
			<td >Bellco Glass, Inc.</td>
			<td >(7910-40150)</td>
			<td >Bioreactor vessel. 1 required.</td>
		</tr>
		<tr >
			<td >Six port assembly</td>
			<td >Bellco Glass, Inc.</td>
			<td >Custom&#160;</td>
			<td >For the bioreactor vessel. Tubing Specs: .125&#34; OD x .055&#34;ID. Port A: 1.0&#34; long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0&#34; long above cap slug, 33 mm long below. Port D: 1.0&#34; long above cap slug, 65 mm&#160; long below. 1 required. Includes 45 mm diameter polypropylene open top screw cap and a white silicone gasket to ensure a tight seal between the cap and the vessel.&#160;</td>
		</tr>
		<tr >
			<td >Scotch Magic Tape 3105, 3/4 x 300 Inches, Pack of 3</td>
			<td >Amazon</td>
			<td >B0009F3P3U</td>
			<td >Clear scotch tape. This is available from many other vendors. It is used to cover markings on the culturing vessel and to secure the coverglass with the PDMS channel to the aluminum support frame.</td>
		</tr>
		<tr >
			<td >1/16&#34; ID x 3/16&#34; OD x 1/16&#34; Wall Tygon Sanitary Silicone Tubing</td>
			<td >United States Plastic Corp.</td>
			<td >57288</td>
			<td >Tubing. ~25&#39; required.</td>
		</tr>
		<tr >
			<td >Cole-Parmer Twistit white rubber stopper, size 10</td>
			<td >Cole-Parmer</td>
			<td >EW-62992-32</td>
			<td >Media flask stopper and effluent flask stopper. 2 required.</td>
		</tr>
		<tr >
			<td >2L Laboratory Flask</td>
			<td >Pyrex</td>
			<td >4980</td>
			<td >Media flask and effluent flask. 2 required.</td>
		</tr>
		<tr >
			<td >Day pinchcock</td>
			<td >Fisher Scientific</td>
			<td >5867</td>
			<td >For pinching tubes shut. 3 required.</td>
		</tr>
		<tr >
			<td >Replacement tubing assembly 1/16&#34; ID</td>
			<td >Traceable Products</td>
			<td >3372</td>
			<td >The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td>
		</tr>
		<tr >
			<td >Replacement tubing assembly 1/50&#34; ID</td>
			<td >Traceable Products</td>
			<td >3371</td>
			<td >The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td>
		</tr>
		<tr >
			<td >Male luer with lock ring x 1/16&#34; hose barb, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-00</td>
			<td >Connectors. ~10 luers are required.</td>
		</tr>
		<tr >
			<td >Male luer with lock ring x 1/8&#34; hose barb, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-04</td>
			<td >Connectors. 5 required, one for each rubber stopper hole to fill with tubing.</td>
		</tr>
		<tr >
			<td >Female luer x 1/16&#34; hose barb adapter, Nylon, 25/pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45502-00</td>
			<td >Connectors. ~10 luers required.</td>
		</tr>
		<tr >
			<td >Female luer x 3/16&#34; hose barb adapter</td>
			<td >Cole-Parmer</td>
			<td >EW-45502-08</td>
			<td >Connectors. ~10 luers required.</td>
		</tr>
		<tr >
			<td >Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td>
			<td >Cole-Parmer</td>
			<td >SC-45502-28</td>
			<td ></td>
		</tr>
		<tr >
			<td >Cole-Parmer Luer Accessory, Male Luer Lock Plug, Nylon, 25/Pk</td>
			<td >Cole-Parmer</td>
			<td >EW-45505-56</td>
			<td ></td>
		</tr>
		<tr >
			<td >Microbore PTFE Tubing, 0.022&#34;ID x 0.042&#34;OD, 100 ft/roll</td>
			<td >Cole-Parmer</td>
			<td >EW-06417-21</td>
			<td >Tubing. 1 roll required.</td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone tubing, L/S 13, 25 ft</td>
			<td >Cole-Parmer</td>
			<td >EW-96410-13</td>
			<td >Tubing. ~25&#39; required.</td>
		</tr>
		<tr >
			<td >3/16&#34; ID x 1/4&#34; OD x 1/32&#34; Wall Tygon Sanitary Silicone Tubing</td>
			<td >United States Plastic Corp.</td>
			<td >57293</td>
			<td >Tubing. ~1&#39; required.</td>
		</tr>
		<tr >
			<td >Vacuum filter</td>
			<td >Fisher Scientific</td>
			<td >974107</td>
			<td >Nalgene vacuum filter for sterile filtering media.</td>
		</tr>
		<tr >
			<td >Aquel Oxy-Boost 200</td>
			<td >Rena Aquatic Supply</td>
			<td >AP200</td>
			<td >Dual diaphram adjustable flow air pump for aerating and mixing media. 1 required.&#160;</td>
		</tr>
		<tr >
			<td >0.2 &#956;m pore syringe filter</td>
			<td >Corning International</td>
			<td >431229</td>
			<td >This ensures that air from the aquarium pump does not contaminate the apparatus. 1 required.</td>
		</tr>
		<tr >
			<td >Slygard 184 Silicone Elastomer Kit</td>
			<td >Dow Corning</td>
			<td >Slygard 184</td>
			<td >For microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >American Safety Razor GEM Scientific Single-Edge Razor Blades</td>
			<td >Fisher Scientific</td>
			<td >17989000</td>
			<td >For cutting tubes and PDMS. 1 blade required.</td>
		</tr>
		<tr >
			<td >Harris Uni-Core hole puncher 1.2mm ID</td>
			<td >Sigma-Aldrich</td>
			<td >WHAWB100028 ALDRICH</td>
			<td >For punching inlet/outlet in microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >Microscope cover glass 22x60-1.5</td>
			<td >Fisher Scientific</td>
			<td >12-544-G</td>
			<td >For microfluidic device. 1 required.</td>
		</tr>
		<tr >
			<td >Rectangular aluminum frame with a square window</td>
			<td >Custom</td>
			<td >Custom</td>
			<td >To support the microfluidic channel. Outer dimensions: 3 inches x 1.25 inches. 
			Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches 
			Thickness: ~1/32 inches</td>
		</tr>
	</tbody>
</table>

design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications

光遺伝学システムは、細胞プロセスを変更するために特定の波長の光に反応して立体構造を変更遺伝的にコードされたタンパク質を利用します。光遺伝学システムのプログラムされた照明および刺激を組み込んだシステムを培養し、測定が必要です。我々が構築した光のプログラムされた用量で微生物細胞を照射し、自動的に取得し、流出液中の細胞の画像を分析するために、連続培養装置を使用するためのプロトコルを提示します。ケモスタット、この装置の動作は、増殖速度と細胞環境を厳密に制御することを可能にします。連続的な細胞培養の流出物は定期的にサンプリングし、細胞をマルチチャンネル顕微鏡によって画像化されます。蛍光強度および培養流出物から採取した細胞の細胞形態の動的応答が複数日にわたって測定されるように培養、サンプリング、画像化、および画像解析は完全に自動化されていますユーザーの入力なし。我々は、動的に転写を活性化する光遺伝学系で操作サッカロマイセス・セレビシエの株においてタンパク質産生を誘導することによって、この培養装置の有用性を実証します。

この装置は、CRY2 / CIB1タンパク質対30に基づいて、誘導光遺伝学転写系を介して青色光に応答して、黄色蛍光タンパク質（YFP）を発現するS.セレビシエの培養物を刺激するために使用しました。細胞は0.2±0.008の平均希釈率でリン酸限定培地でchemostatically成長させました。リン酸塩の制限は、一般に、成長速度を制御するためにS.セレビシエ</e...

Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

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

我々は、倒立顕微鏡を用いて流出液中の微生物、定期的に画像を細胞の培養物を照明する光遺伝学システムを使用するための連続的な培養装置を設計しました。照明の動的応答は、数日間にわたって測定することができるように培養、サンプリング、画像化、および画像解析は完全に自動化されています。

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 ...

design-implementation-an-automated-illuminating-culturing-sampling

Research

JoVE Journal

Bioengineering

9.6K ビュー.  University of Wisconsin-Madison. 我々は、倒立顕微鏡を用いて流出液中の微生物、定期的に画像を細胞の培養物を照明する光遺伝学システムを使用するための連続的な培養装置を設計しました。照明の動的応答は、数日間にわたって測定することができるように培養、サンプリング、画像化、および画像解析は完全に自動化されています。 

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

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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	 </div>

GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

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 ...

生物工学

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

用絹フィルム培養システム in vitroで分析とマテリアルデザイン

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

低侵襲手術でのソフトモジュラーロボットのエラストマーユニットの設計と製作

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高スループットの自動モデル IgG1 CHO 細胞での生産のための Microbioreactor システム

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

緑膿菌自動の高スループット マイクロ流体システムを使用して上のせん断応力のライブ細胞解析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

自動化された微生物叢システムを用いた中国ハムスター卵巣細胞からのモノクローナル抗体の精製と分析

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Design and Analysis for Fall Detection System Simplification

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system that can be easily implemented and adopted. The methodology is based on the configuration of specific types of sensors, machine-learning methods and procedures. The protocol is divided into four phases: (1) database creation (2) data analysis (3) system simplification and (4) evaluation. Using this methodology, we created a multimodal database for fall detection and human activity recognition, namely UP-Fall Detection. It comprises data samples from 17 subjects that perform 5 types of falls and 6 different simple activities, during 3 trials. All information was gathered using 5 wearable sensors (tri-axis accelerometer, gyroscope and light intensity), 1 electroencephalograph helmet, 6 infrared sensors as ambient sensors, and 2 cameras in lateral and front viewpoints. The proposed novel methodology adds some important stages to perform a deep analysis of the following design issues in order to simplify a fall detection system: a) select which sensors or combination of sensors are to be used in a simple fall detection system, b) determine the best placement of the sources of information, and c) select the most suitable machine learning classification method for fall and human activity detection and recognition. Even though some multimodal approaches reported in literature only focus on one or two of the above-mentioned issues, our methodology allows simultaneously solving these three design problems related to a human fall and activity detection and recognition system.

We present a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition system. The goal is to build a system for accurate fall detection that can be easily implemented and adopted.

落下検知システムの単純化のための設計と解析

This paper presents a methodology based on multimodal sensors to configure a simple, comfortable and fast fall detection and human activity recognition ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

小規模セル処理のための自動カウンターフロー遠心システム

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...