Name: design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications
Uploaded: 2017-02-19T14:00:00
Description: Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

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。

<img alt="図1" src="/files/ftp_upload/54894/54894fig1.jpg" /> 図1：連続培養装置。この略図は、それを培養するために使用されたときに装置を組み立てすべきかを示し照明、及び微生物の光学的特性を測定します。 <a href="http://ecsource.jove.com/files/ftp_upload/54894/54894fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <p class...

<ol>
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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. 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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. 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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. 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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>

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 ...

The overall goal of this procedure is to assemble a continuous culturing vessel, in which the response to illumination of optogenetic microbes can be measured with a microscope automatically in real time and over multiple days. This method can help answer key questions in cellular biology and metabolic engineering, such as how dynamic gene expression can elicit responses which differ from static gene expression. The main advantage of this method it that continuous culture enables measurements to be collected for multiple days.To begin, solder the ground line, the data line, and the positive voltage line of the digital thermometer to the printed circuit board. Clip off one pin from a female three pin header and trim the remaining two pins. Solder this in the pair of holes labeled R2, and connect the two soldered pins by inserting a 4.7 kilo ohm resistor in the pin header.Next connect the remaining components to the circuit board. Overlay the circuit board on to the micro controller. Set up the light proof enclosure, and install all software as described in the accompanying text protocol.First, place the long end of the aluminum port through the gasket into the culturing vessel and screw on the cap. Cover one short aluminum port outlet with medium diameter silicone tubing. Plug the distill end by inserting a female luer lock, and then connecting a male luer lock plug.This outlet is supplementary and will not be used. Connect two short segments of medium diameter silicone tubing with male and female luer locks. Then connect one end to a short aluminum port outlet, and plug the distill end.The vessel will be inoculated through this tube. Next, connect two medium tubes to the ends of the medium diameter peristaltic tubing and connectors. Connect one end to a short aluminum port, and plug the other.Later, this will be connected to the media flask. Connect a medium diameter silicone tube to the longest aluminum port. The tube should be long enough to reach the media flask.Connect another short segment a few inches long to the end, and then insert it into the rubber stopper on the media flask. Clamp the tube. When later unclamped, this connection allows the culture to be mixed, aerated, and kept at a positive pressure by the incoming bubbles.Insert a medium diameter tube long enough to reach the bottom of the flask into the stopper through which media will be transferred. Connect another medium diameter tube to this one, and then a wide diameter tube on the end. Plug the distill end.Fill the third hole in the stopper with a short segment of medium diameter tubing, connected to two other segments of medium diameter tubing, and plugged at the distill end. This will be connected to a vacuum pump, and later to an aquarium pump. Insert a male luer lock into a segment of narrow diameter silicone tubing and then firmly insert PTFE tubing.Then attach a female luer lock to a medium diameter tube. Thread the female lock along the PTFE tube to connect the locks and the other to a short aluminum port. Ensure that the PTFE tube inside the culturing vessel dangles below the second longest aluminum tube, and above the bottom.Trim as needed. Media will be sampled from here to the imaging channel, and the narrow tubing minimizes the intermediate volume. Next, connect the exposed narrow silicone tube to the narrow peristaltic tube and connectors.To this connect three segments of narrow diameter tubing and two segments of PTFE tubing, alternating them. Connect this to the effluent flask, via medium diameter tubing. Then connect one end of a medium diameter silicone tube to the second longest tube of the aluminum port in the other end of the effluent flask.This aluminum tube sets the culture volume, and excess culture overflows into the effluent container. Wrap the lids of the flask with aluminum foil, and then autoclave the assembly for 30 minutes at 121 degrees Celsius and 15 PSI. Attach the vacuum filter to a 100 milliliter bottle.Remove the nipple cover, and then remove the white plug from the nipple of the vacuum filter with sterile tweezers. Connect the wide silicone tube to the nipple, the media flask's other free tube to a vacuum pump, and ensure that the third tube connecting the media flask to the culturing vessel is clamped shut. Fill the filter with media, turn on the vacuum pump, and then filter the rest of the media.Clamp the medium silicone tubing connected to the vacuum and turn off the vacuum pump. Remove the luers from the intermediate segment of medium silicone tubing connected to the vacuum pump and insert the blue end of a sterile syringe air filter into this tube. Then clamp the wide silicone tube and disconnect the male luer from it.Disconnect the plug from the female luer of the culturing vessel's media inlet tube. Connect these luers to enable media to be pumped into the culturing vessel. Then, remove the filter from the 100 milliliter bottle, and attach the bottle cap.Use this media to prepare a starter culture. Set the continuous culturing assembly near a microscope with the media flask higher than the culturing vessel, and the effluent flask lower than the culturing vessel to prevent back flow. Securely tape down the rubber stoppers on the media and effluent flasks.Next unplug the ends of the PTFE tube from the narrow silicone tube connecting them and plug these ends into the inlet and outlet of a prepared microfluidic device. Then push media into the culturing vessel, by connecting the white end of the syringe air filter to the aquarium pump. When media reaches the level of the effluent port wrap the medium media inlet tubing and connectors around the slow peristaltic pump, and the narrow sample outlet tubing and connectors around the fast peristaltic pump.Aerate the media by unclamping the air tube between the media flask and culturing vessel. Then, tape the heating pad and thermometer to the culturing vessel so that it's temperature can be controlled. Coil the media tubing around the culturing vessel, so the entering media will be at the same temperature as the vessel.Then insert the culturing vessel into the black foam enclosure over the LED matrix and ensure that the tubes are not pinched. Use the bioreactor controller plugin to set the slow media pump on for one tenth of every 30 second interval. The flow rate will be very low, but greater than the rate of evaporation.Clamp the incoming air tube between the media flask and culturing vessel. Use a pipette to inoculate the culturing vessel with one milliliter from a starter culture. Then clamp the distill end of this tube, and unclamp the air tube.Let the culture grow overnight. Cover the enclosure so that no light enters. The next day, use the bioreactor controller plugin to set the peristaltic pumps to rotate for a greater proportion of time, corresponding to the desired media and sampling flow rates.Let the culture density equilibrate overnight. The next day fill the stage position list and micro manager with a set of non overlapping positions where cells pumped into the microfluidic channel will be in the focal plane. Then open the bio reactor controller plugin.Select the desired LED matrix time course, imaging channels, and other experimental settings from the prompts, and then begin collecting data. This apparatus was used to stimulate a culture of saccharomyces cerevisiae, expressing yellow fluorescent protein in response to blue light via an inducible optogenetic transcription system. Base contrast and fluorescent images of the microfluidic channel were acquired.Cell outlines were identified from the phase contrast images, mapped to the fluorescent images, and the fluorescent images were recorded in real time. The heat map here shows the fluorescence distribution of a culture due to yellow fluorescent protein production upon activation of the optogenetic system. The fluorescence of approximately of 170, 000 cells was analyzed from over 30, 000 images, acquired from 28 stage positions over 70 hours.During this study, the culture was exposed to varying intensities of blue light for six hour intervals, followed by six hours of complete darkness. Cells had been exposed to light prior to the first measurement which is why it's fluorescence intensity is decreasing during the first dark period. Once mastered, this technique can be done in eight hours, over the course of three days, if it is performed properly.While attempting this procedure, it's very important to use sterile technique. Following this procedure, other methods, like real time feedback control, can be used to control protein concentration. This technique will enable organisms with optogenetic constructs to be studied dynamically, over longer periods of time, longer than what is possible using plates or batch culture.After watching this video, you should have a good understanding of how to integrate a continuous culturing apparatus, a light, and a microscope, to stimulate and study optogenetic microbes.

Research

JoVE Journal

Bioengineering

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk Film Culture System for in vitro Analysis and Biomaterial Design

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 ...

生物工学

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 ...

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

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

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; ...

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

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

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

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

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

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 ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

光遺伝学的実験のための簡便で汎用性の高い照明システムの構築

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...