Name: design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications
Uploaded: 2017-02-19T14:00:00.000+00:00
Duration: 11 min 13 s
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

Biz yararlı tartışmalar ve düzenleme için protokol, Kieran Sweeney test yardım için Molly Lazar ve Verónica Delgado kabul etmek ister ve yazının eleştirel okuma Taylor Scott, My An-adirekkun ve Stephanie Geller olacaktır. Megan Nicole McClean, Ph.D. Burroughs Wellcome Fonu Bilimsel Arayüz bir Kariyer Ödülü sahibidir.

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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. 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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. 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Yazarlar ifşa hiçbir şey yok.

Biz esneklik düşünülerek bu cihazı tasarladı. Kullanılan tüm kod ücretsiz ve açık kaynak. Segment hücrelere varsayılan görüntü analizi süreci basit ve hızlı bir şekilde çalışır. Özel analizi, FIJI grafik kullanıcı arayüzü ile temsili bir görüntüsünü analiz eden bir Beanshell komut girişini dönüştürme ve sonra komut dosyasını çağırmak için eklenti ayarlarken kullanıcı girişi kayıt ile uygulanabilir. o çağrıldığında, bu komut arka plan çıkarılır görüntülerin en...

Optogenetic sistemleri gen ekspresyonu, 1, 2, 3, 4, 5 protein lokalizasyonu, 6 protein aktivitesini, 6, 7, 8 protein bağlama, 8, 9, 10 ve protein degradasyonunu da dahil olmak üzere hücresel süreçleri artan listesini kontrol etmek için ışık kullanır. Programlı optik stimülasyon ile kontrollü bir ortamda hücrelerin kültüre ve biyolojik ilgili zaman ölçekleri üzerindeki tepkisini ölçmek için 11 bir yöntem hücre biyolojisi ve biyoteknoloji araştırma için bu araçların potansiyelini gereklidir. Önerilen yöntem, AE, iyi karışmış sürekli bir hücre büyüme hızını sabitlemek için chemostasis yararlanırpuan ve programlanan aydınlatma maruz sıcaklık kontrollü cam kültürleme kabı 12, 13. bir inverted mikroskop ile kültür atık suyunda görsele tek tek hücreler programlanmış aydınlatma kültür tepkisini ölçmek için. floresan yoğunluğu ve atık hücre kültürünün hücresel morfoloji kullanıcı girişi olmadan birden fazla gün içinde ölçülebilir, böylece kültür, örnekleme, görüntüleme ve görüntü analizi tam otomatik vardır. Bu protokol, büyüyen hücre kültürü ve mikroskopi aşina en laboratuvarlarında uygulanabilir ve kullanılan cihaz ucuz ve kolaylıkla temin edilebilen bileşenler yapılmıştır. Şeffaf bir kültürleme kabı ışık 1 uW / cm2 -10 mW / cm2 yayabilen ışık yayan diyotlar (LED), bir matris üzerine yerleştirilir. Mikroplar sürekli kültür kabında yetiştirilir; bir peristaltik pompa de medya eklemek için kullanılırseyrelme oranı, bir mikroskop daha az bir oranda kültür çekmek için kullanılır ve fark, taşma çıkışı kaçar. Bir ısıtma yastığı sıcaklığını koruyor. Hava sürekli pozitif basıncı muhafaza etmek ve karıştırmak ve havalandırmak için kültür kültür kabına pompalanır. Hava pompası dışında, bu cihazlar için güç aynı zamanda bir termometre ve bağlı masaüstü bilgisayardan giriş alır bir mikro denetleyici tarafından düzenlenir. atık hücre kültürü ters bir mikroskop sahnede bir mikroakışkan cihaz pompalanır. Floresan olmayan ve floresan görüntüler otomatik olarak elde edilir. Görüntülerde hücreler ilgi (ROI) bir bölge olarak her bir hücreyi bulur ve her ROI özelliklerini ölçen bir algoritma ile karakterizedir. Bu protokolün bir uygulama göstermek için, bir mavi ışık responsi ile mühendislik Saccharomyces cerevisiae hücreleri değişen ışık şiddetlerinde yanıt ölçülenfloresan protein transkripsiyonunu kontrol eden Optogenetic Sistemi. Bu sistemde gen ekspresyonunu kontrol etmek için çok sayıda Optogenetic sistemleri zaten 14, 15, 16 bulunduğu için yaygın olarak ekmek mayası olarak bilinen, S. cerevisiae, seçilmiştir. Bundan başka, bu model organizma yaygın sistem biyolojisi 17 ve biyo teknolojik uygulamalarda 18, 19, 20 için bir şasi gibi çalışmaları için kullanılır. Bizim Örnek Sonuçlar Bu protokol, giriş ışık değişen yoğunluklarda ve flüoresan raportör üretimini ölçerek çok gün boyunca kültürü transkripsiyonunu kontrol etmek için kullanılabileceğini gösterir.

<table><tbody><tr><td>Extensive lab manual</td><td>GitHub</td><td>NA</td><td>An extensive, regularly updated lab manual is available in the &amp;ldquo;Optogenetic Chemostat Files&amp;rdquo; 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 &quot;http://fritzing.org/download/&quot;</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&quot; 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 &amp;mu;F capacitor</td><td>SparkFun Electronics</td><td>COM-08375</td><td>Stabilizes voltage in PCB. 1 required.</td></tr><tr><td>10 &amp;mu;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&quot;)</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.&amp;nbsp;</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&quot; (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>&amp;nbsp;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 &quot;https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release&quot;</td></tr><tr><td>FIJI</td><td>ImageJ</td><td>NA</td><td>The free, open-sourced image analysis software is available at &quot;http://fiji.sc/&quot;</td></tr><tr><td>Arduino Integrated Development Environment</td><td>Arduino</td><td>NA</td><td>The free, open-sourced IDE is available at &quot;https://www.arduino.cc/en/Main/Software&quot;</td></tr><tr><td>Custom code</td><td>GitHub</td><td>NA</td><td>The custom microcontroller code and &quot;Bioreactor Controller&quot; plugin are available in the &amp;ldquo;Optogenetic Chemostat Files&amp;rdquo; 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 &amp;ldquo;Optogenetic Chemostat Files&amp;rdquo; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td></tr><tr><td>Tota-frost gels (diffusion paper)</td><td>B&amp;amp;H</td><td>B&amp;amp;H # LOFSFTL&lt;br/&gt; 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&amp;nbsp;</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&quot; 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&amp;nbsp;</td><td>For the bioreactor vessel. Tubing Specs: .125&quot; OD x .055&quot;ID. Port A: 1.0&quot; long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0&quot; long above cap slug, 33 mm long below. Port D: 1.0&quot; long above cap slug, 65 mm&amp;nbsp; 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.&amp;nbsp;</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&quot; ID x 3/16&quot; OD x 1/16&quot; Wall Tygon Sanitary Silicone Tubing</td><td>United States Plastic Corp.</td><td>57288</td><td>Tubing. ~25&#039; 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&quot; 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&quot; 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&quot; 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&quot; 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&quot; 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&quot; 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&quot;ID x 0.042&quot;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&#039; required.</td></tr><tr><td>3/16&quot; ID x 1/4&quot; OD x 1/32&quot; Wall Tygon Sanitary Silicone Tubing</td><td>United States Plastic Corp.</td><td>57293</td><td>Tubing. ~1&#039; 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.&amp;nbsp;</td></tr><tr><td>0.2 &amp;mu;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.&lt;br/&gt; Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches&lt;br/&gt; Thickness: ~1/32 inches</td></tr></tbody></table>

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

Optogenetic sistemleri hücresel prosesleri değiştirebilir ve ışık spesifik dalga boylarında yanıt olarak şekil değiştirirler genetik olarak kodlanmış proteinleri kullanılmaktadır. Optogenetic sistemlerin aydınlatma ve stimülasyon programlanan kullanan sistemler kültürlenmesi ve ölçülmesi için bir ihtiyaç vardır. Biz bina ve ışık programlanmış dozlarda mikrobiyal hücrelerin aydınlatmak ve otomatik olarak kazanmak ve atık hücrelerin görüntülerini analiz etmek için sürekli bir kültür cihazı kullanılarak bir protokol mevcut. Bir kemostat bu düzeneğin çalışması büyüme ve hücre ortamı sıkı bir şekilde kontrol edilmesini sağlar. sürekli hücre kültürü atık düzenli örneklenmiş ve hücreler çok kanallı mikroskobu ile görüntülenmiş. floresan yoğunluğu ve kültür mahsulünden örneklenen hücrelerin hücresel morfoloji dinamik tepkiler birden fazla gün boyunca ölçülen böylece kültür, örnekleme, görüntüleme ve görüntü analizi tam otomatik olankullanıcı girişi olmadan. Dinamik olarak transkripsiyonu aktive bir Optogenetic sistemi ile işlenmiş, Saccharomyces cerevisiae protein üretimini indükleyen bu kültürleme cihazının kullanımını göstermektedir.

Bu aygıt, cry2 / CIB1 proteini çifti 30 göre, indüklenebilir kopyalama Optogenetic sistemi ile mavi ışığa karşılık olarak sarı floresan protein (YFP) ifade S. cerevisiae&#39;nin kültürü teşvik etmek için kullanıldı. Hücreler 0.2 ± 0.008 ortalama seyrelme oranı ile fosfat sınırlı medyada chemostatically yetiştirilmiştir. Fosfat sınırlama genel olarak büyüme oranını kontrol etmek için, S. cerevisiae kemostat deneyl...

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

Biz bir inverted mikroskop ile atık mikropların kültürler ve düzenli görüntü hücrelere aydınlatmak için Optogenetic sistemleri ile kullanım için sürekli bir kültür cihazı tasarlanmıştır. aydınlatma dinamik tepkiler birkaç gün içinde ölçülebilir, böylece kültür, örnekleme, görüntüleme ve görüntü analizi tam otomatik vardır.

Mikrobiyal optogenetic Uygulamaları Tasarım ve Otomatik Aydınlatıcı, kültürleme ve Örnekleme Sistemi Uygulaması

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 Görüntüleme.  University of Wisconsin-Madison.  Biz bir inverted mikroskop ile atık mikropların kültürler ve düzenli görüntü hücrelere aydınlatmak için Optogenetic sistemleri ile kullanım için sürekli bir kültür cihazı tasarlanmıştır. aydınlatma dinamik tepkiler birkaç gün içinde ölçülebilir, böylece kültür, örnekleme, görüntüleme ve görüntü analizi tam otomatik vardır. 

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

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Bir Optogenetic kontrol etmek ve hücre-hücre etkileşimleri gen ifade desenleri analiz yöntemi

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetik

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Kemirgen Santral Sinir Sistemi vivo optogenetic Stimülasyon içinde

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Nörobilim

Zebra Balıklarında Optogenetik Sinyal Manipülasyonu ile Gelişimin İncelenmesi

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Yazar Spotlight: Hücre Kaderi Kararlarını Çözmek için Zebra Balığı Embriyolarında Sinyalizasyonu Manipüle Etmek 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Zebra Balığı Embriyolarında Optogenetik Sinyal Aktivasyonu

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Gelişim Biyolojisi

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Optogenetik Deneyler için Basit ve Çok Yönlü Bir Aydınlatma Sistemi Oluşturma

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

Biyomühendislik

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Memeli Hücrelerinde Kararlı Optogenetik Gen Devrelerinin Güvenilir Mühendisliği ve Kontrolü

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Mikrobiyal Kimyasal ve Protein Üretimi için Işık Kontrollü Fermantasyonlar

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

İnsan Nöromüsküler Kavşağının Optogenetik Modelinin Mühendisliği ve Karakterizasyonu

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Lustro Otomasyon Tekniği Kullanılarak Maya Optogenetik Yanıt Analizi

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems.
Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of&#160;their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro&#39;s components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Yazar Spotlight: Lustro Kullanarak Optogenetik Araştırmalarını İlerletmek 

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast.

Otomatik platform Lustro kullanılarak mayada yüksek verimli optogenetik deneyler

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to ...

Mikrobiyal optogenetic Uygulamaları Tasarım ve Otomatik Aydınlatıcı, kültürleme ve Örnekleme Sistemi Uygulaması

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