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| | <img src="https://static.igem.org/mediawiki/2018/e/e5/T--NUS_Singapore-A--Hardware_header_C.png" class="header"> | | <img src="https://static.igem.org/mediawiki/2018/e/e5/T--NUS_Singapore-A--Hardware_header_C.png" class="header"> |
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| − | <h1>Introduction</h1> | + | <h1>Fermentation Chamber (INNOVATION)</h1> |
| | <br> | | <br> |
| − | | + | <looping gif></looping> |
| − | <p>Our hardware team developed two sets of hardware to address two problems in synthetic biology, and complement the work of the wet lab team to complete our optogenetic biomanufacturing platform. </p> | + | |
| | <br> | | <br> |
| − | | + | <h2>Function</h2> |
| − | <h2>Problem #1</h2> | + | |
| | <br> | | <br> |
| − | <p>The first problem is that while there is a rapidly-growing interest in using optogenetics for biomanufacturing, development of custom tools to support the research of optogenetic circuits cannot match this pace, and is insufficient to meet user needs<sup>[2]</sup>. An example of the most current hardware tools available is a modified Tecan microplate reader, which provides controlled illumination on top of its usual measurement capabilities<sup>[3]</sup>. Such an approach is costly and requires specialized knowledge of the microplate reader model. Another example would be the open-source light exposure tool constructed for a 24-well plate<sup>[4]</sup>. To our team, it seemed that scaling-up in optogenetic research (Figure 1) was not well-supported by current hardware solutions, which only cater to microwell plates. </p> | + | <p>The fermentation chamber contains the bacterial culture. It also comes with a cover designed to include a means of illuminating the chamber’s contents. </p> |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 1</b>. Scaling-up in optogenetics research - from the microplate to small-scale bioreactor</figcaption>
| + | |
| − | </figure>
| + | |
| − | <br>
| + | |
| − | <p>Yet, the biomanufacturing industry is expected to deliver products to the market, in high volumes, at high quality, and at competitive prices<sup>[5]</sup>. If we are ever to bring our optogenetic biomanufacturing platform to an industrial scale, it is necessary to bridge the gap between the microplate and the industrial bioreactor, and adapt our cells for actual large-scale bioreactor conditions. We thus designed a suite of three devices, called <i>PDF-LA!</i>, which enables the characterization of optogenetic circuits at different scales - 12-well <b><u>P</u></b>late, petri <b><u>D</u></b>ish, and conical <b><u>F</u></b>lask. We also created a bench-top optogenetic bioreactor, <i>Light Wait</i>. It is our vision that these devices will empower optogenetic researchers to make great leaps forward in their research, although we acknowledge that there is a still-greater leap between our humble bioreactor and an industrial bioreactor (Figure 2). For now, it is enough for us to have taken the first few steps.</p>
| + | |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 2</b>. The components in Figure 1 (bottom right-hand corner) are still dwarfed by an industrial bioreactor.</figcaption>
| + | |
| − | </figure>
| + | |
| | <br> | | <br> |
| | | | |
| − | <h2>Problem #2</h2> | + | <h2>Design</h2> |
| − | <p>The second problem is that although a proof-of-concept already exists for optogenetic biomanufacturing, the process can be further optimized to bring the vision of an industrial-scale optogenetic bioreactor closer to reality. </p>
| + | |
| | <br> | | <br> |
| − | <p>For some background, Zhao et al. have increased yield of isobutanol from yeast by using a blue light repressible system in a simple bioreactor, showing the potential of optogenetics in biomanufacturing<sup>[6]</sup>. However, they did not optimize the duration or intensity of blue light, instead shining blue light periodically. We discovered that dynamic regulation is a good method for optimizing biomanufacturing, because prioritization of growth and production can be achieved simultaneously. We distilled this observation from both literature<sup>[7]</sup> and our <a href="#">Human Practice</a> activities. Dynamic regulation can be achieved through computer-assisted feedback control, and we found that Argeitis et. al developed automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth<sup>[8]</sup>. So far this is the most recent and sophisticated feedback system for optogenetics. However after examining his method, we found that while his feedback control system was closed-loop, his physical system was open. Measurement samples were discarded as waste. This is not advantageous to biomanufacturing, as this will lead to much product being wasted, lowering effective yield.</p> | + | <p>We selected components of the fermentation chamber based on whether they were easily obtainable and modifiable. This was because it was difficult to fabricate a cylindrical, watertight chamber using conventional methods of prototyping such as laser cutting and 3D printing. Rather than spend time attempting to manufacture a suitable container from scratch, we looked to modifying existing commercial, easily obtainable products. This also makes it easier for others to make their own bioreactor based on our work.</p> |
| | <br> | | <br> |
| − | <p>
| |
| − | To solve this, we combined the insights and design features from these two systems (Zhao and Argeitis) to create an automated, closed-loop feedback control system for <i>Light Wait</i>.</p>
| |
| | | | |
| − | | + | <h3>Container</h3> |
| − | <h3>PDF-LA!</h3>
| + | |
| − | <br>
| + | |
| − | <h4>Function</h4>
| + | |
| | <br> | | <br> |
| − | <p>Plate-Dish-Flask Light Apparatus (<i>PDF-LA!</i>) supports optogenetic research by allowing researchers to investigate cells cultured in 12-well plates, petri dishes, and Erlenmeyer flasks.</p> | + | <p>We decided on a suitable working volume for our bioreactor based on literature review<sup>[1]</sup>. Possible working volumes suitable for the laboratory ranged from 0.5 L to 2 L. After discussion, we decided that 0.5 L would be a manageable volume. We hypothesized that this volume was sufficient for us to demonstrate proof-of-concept. It is also more portable.</p> |
| | <br> | | <br> |
| | | | |
| − | <h4>Product Demonstration</h4>
| |
| | | | |
| − | <br> | + | <p>A bail jar from IKEA was repurposed for this. Discarding the original glass cover, we retained the rubber ring and two-part wire clasp.</p> |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 2</b>. gif in progress</figcaption>
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 1</b>. Showcase of PDF-LA!</figcaption>
| + | |
| − | </figure> | + | |
| | <br> | | <br> |
| | | | |
| − | <video src="#" width="300"> uploaded to drive </video>
| + | <h3>Cover</h3> |
| − | <figure><figcaption>Video 1. With PDF-LA!, you’ll be light-years ahead of the competition! At the very least, you can program your own snazzy light show and be the envy of other optogenetics researchers.</figcaption></figure>
| + | |
| − | | + | |
| − | <p>The utility and functionality of <i>PDF-LA!</i> was validated by user feedback. We also used it when characterizing the behaviour of EL222 in repressible and inducible systems, thus producing what you see on our Results page.</p>
| + | |
| − | <br>
| + | |
| − | | + | |
| − | <h3>How it Works</h3> | + | |
| − | <br>
| + | |
| − | <h4>Operation</h4>
| + | |
| − | <br>
| + | |
| − | <p>This operation guide assumes that all electronics have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. Instructions may be found on our dedicated page for <a href="#"><i>PDF-LA!</i></a>. </p>
| + | |
| − | <br>
| + | |
| − | <ol>
| + | |
| − | <li>Place your container into the required holder. If using an Erlenmeyer flask, first rest the flask on <i>D-LA!</i>, then place the flask adapter over the flask to form <i>F-LA!</i>. Keeping a firm grip on <i>F-LA!</i>, pull the flask upwards sharply to ensure a tight fit.</li>
| + | |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 2</b>GIF of plate, dish, flask going into each container</figcaption>
| + | |
| − | </figure>
| + | |
| − | <br>
| + | |
| − | <li>Connect the AC adapter to the Arduino and wall socket.</li>
| + | |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 2</b>arduino, AC adapter picture, wall socket picture, arrows to indicate</figcaption>
| + | |
| − | </figure>
| + | |
| − | <br>
| + | |
| − | <li>Turn on the wall switch controlling the AC adapter. </li>
| + | |
| − | <br>
| + | |
| − | </ol>
| + | |
| | <br> | | <br> |
| − | <p>The devices should light up as shown in the product demonstration video above.</p> | + | <p>We opted not to use the original cover because we knew we would have to perforate it to accommodate tubing. Drilling would generate many points of failure. For example, the glass cover might shatter from the stress, it is challenging to control the accuracy of our drilling, and drilling may not be able to provide the required precision.</p> |
| | <br> | | <br> |
| | | | |
| − | <h4>Possible Configurations</h4> | + | <p>We thus retrofitted our own lasercut Plexiglass cover. This allowed us greater flexibility to explore potential sensing modules and components we intended to add to our system. Considered, but eventually discarded, were modules like a pH meter, as we decided to focus on other metrics to indicate cell stress (See <a href="#">Design: Stress Reporter</a>). Eventually, we decided to have 4 small holes, through which acrylic/glass tubes with outer diameters of 6 mm should be inserted, and 2 large holes, to each fit a test tube (Figure 1).</p> |
| | <br> | | <br> |
| − | <p><i>DF-LA!</i> was designed with modularity and flexibility as fundamental guiding principles. Many configurations are possible, enabling researchers to customize their experimental setups to a greater degree. While P-LA! was designed separately and thus does not have this functionality, a final solution, <a href="#"><i>PDF-LA! 2.0</i></a>, to provide a truly integrated solution was designed and can be found on our dedicated page for <a href="#"><i>PDF-LA!</i></a>. Unfortunately, while we could not actualize this solution due to time constraints and limits on our 3D printing equipment, it is our hope that future iGEM teams may be able to experience, test, and improve <i>PDF-LA! 2.0</i>’s utility and functionality.</p> | + | <p>For the acrylic tubes, 2 tubes would be connected to a peristaltic pump, one to an air pump, and the last one to a length of silicone tubing so that new media can be introduced. The test tubes act as containers for LEDs, allowing them to suffuse the culture with light (Figure 2).</p> |
| | <br> | | <br> |
| | + | <figure class="figures2"> |
| | + | <img src="#"> |
| | + | <figcaption>Figure 1. Top view of lasercut cover, showing the locations of apertures for acrylic tubes and test tubes.</figcaption> |
| | | | |
| − | <p>Examples of the possible configurations can be found below.</p>
| + | <img src="#"> |
| − | <br>
| + | <figcaption>Figure 2. Section view of fermentation chamber, showing how test tubes can provide an illumination solution. Also included is an example of how an acrylic tube may be inserted into the cover. Tubing and wiring have been omitted for clarity.</figcaption> |
| − | <ul>
| + | |
| − | <li>1 x D-LA!, bottom illu, bottom and top illu</li>
| + | |
| − | <li>1 x F-LA!, bottom illu, bottom and top illu</li>
| + | |
| − | <li>N x DF-LA!, bottom-bottom and bottom and top illu</li>
| + | |
| − | </ul>
| + | |
| − | <br>
| + | |
| − | <h4>Components</h4>
| + | |
| − | <br>
| + | |
| − | <p><i>P-LA!</i> comprises a tech holder and a lighting plate</p>
| + | |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="# " alt="Video"> | + | |
| − | <figcaption><b>Figure 2</b>picture of tech holder, picture of lighting plate, GIF of collapsed assembled <i>P-LA!</i></figcaption>
| + | |
| | </figure> | | </figure> |
| | <br> | | <br> |
| | | | |
| − | <p>Collectively, a single unit of <i>DF-LA!</i> comprises a tech holder, a petri dish illumination column, and a flask adapter. </p> | + | <h3>Stirring</h3> |
| | <br> | | <br> |
| − | <figure class="figures"> | + | <p>The ambient temperature of where our bioreactor would be located was too cold to be conducive for growing bacteria. Another problem we faced was the magnetic stirrer limiting the depth to which the test tubes could be sunken and thus the light penetration. We had a eureka moment when we realized that both problems could be solved by placing Light Wait into a shaking incubator as shown in Light Wait: Product Demonstration.</p> |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 2</b>picture of tech holder, a petri dish illumination column, and a flask adapter, GIF of collapsed assembled DF-LA!</figcaption>
| + | |
| − | </figure>
| + | |
| − | <br>
| + | |
| − | | + | |
| − | | + | |
| − | <p>Presenting, <i>PDF-LA!</i> Click <a href="#">here</a> for its dedicated page.</p>
| + | |
| − | | + | |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure 2</b><i>P-LA!</i> and <i>DF-LA!</i> side by side</figcaption>
| + | |
| − | </figure>
| + | |
| | <br> | | <br> |
| | <hr> | | <hr> |
| | <br> | | <br> |
| − | | + | <h2>Construction</h2> |
| − | <h3>Light Wait</h3> | + | |
| | <br> | | <br> |
| − | <h4>Function</h4>
| + | <p>Do you like cookies? Do you like jars? Your answer doesn’t matter. Make our cover anyway! Batteries not included.</p> |
| − | <p><i>Light Wait</i> supports optogenetic research, especially in optogenetic biomanufacturing, by allowing researchers to scale up to a 500 ml working volume bioreactor.</p> | + | |
| − | <br>
| + | |
| − | <h4>Product Demonstration</h4>
| + | |
| − | <br>
| + | |
| − | <video src="#" width:"300">Bioreactor Backup Video</video>
| + | |
| − | <br>
| + | |
| − | <figure><figcaption><b>Video 2</b>. <i>Light Wait</i> may be housed in a shaking incubator unit such as the one shown above.</figcaption></figure>
| + | |
| | <br> | | <br> |
| | | | |
| − | <h4>Validation</h4> | + | <h3>Bill of Materials</h3> |
| | <br> | | <br> |
| − | <p><i>Light Wait</i> was validated through a series of experiments which first proved each component’s functionality, and then the functionality of the whole system when all the components were assembled. </p> | + | <ul> |
| | + | <li>180 x 100 x 5 mm acrylic sheet x 1</li> |
| | + | <li>Arduino Uno x 1</li> |
| | + | <li>472 nm LED x 16</li> |
| | + | <li>LED driver x 1</li> |
| | + | <li>2 kΩ resistor x 1</li> |
| | + | <li>0.1 uF ceramic capacitor x 1</li> |
| | + | <li>4.7uF electrolytic capacitor x 1</li> |
| | + | <li>28-pin IC socket x 1</li> |
| | + | <li>Test tube x 2</li> |
| | + | <li>AC Adapter x 1</li> |
| | + | </ul> |
| | <br> | | <br> |
| − | <h4>Experimental Plans</h4>
| |
| − | <br>
| |
| − | <h4>Experimental Results</h4>
| |
| − | <br>
| |
| − | <h4>How it Works</h4>
| |
| − | <br>
| |
| − | <p>This operation guide assumes that all components have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. For instructions on how to set up and operate each component of <i>Light Wait</i>, please refer to our dedicated component pages.</p>
| |
| − | <br>
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| − |
| |
| − | <h4>Operation</h4>
| |
| − | <br>
| |
| − | <ol>
| |
| − | <li>Place <i>Light Wait</i> in a shaking incubator unit as shown in our Product Demonstration. Take care to ensure that all wires and tubing are slack and of sufficient length, else they may become disconnected during operation. </li>
| |
| − | <li>Fill and cover the fermentation chamber. </li>
| |
| − | <li>Connect the pump, 2-in-1 sensor, and the fermentation chamber with the silicone tubings in a loop as shown below (Figure _). The remaining 2 small tubes are for introducing more media, and an air pump. </li>
| |
| | | | |
| | + | <h3>Structural Assembly</h3> |
| | <br> | | <br> |
| − | <figure class="figures"> | + | <figure class="figures2"> |
| − | <img src="# " alt="Video"> | + | <img src="#"> |
| − | <figcaption><b>Figure __</b>. Illustration of how the pump, sensor, and fermentation chamber should be connected by silicone tubing.</figcaption>
| + | <figcaption>Figure 3. Fermentation Chamber assembly.</figcaption> |
| | </figure> | | </figure> |
| | <br> | | <br> |
| | | | |
| − | <li>Turn on the AC adapters for the pump and the LEDs in the fermentation chamber.</li> | + | < p> Files for lasercutting are available in SLDPRT format so that you may easily modify its dimensions to fit your jar (please find your own). You may download them all as a ZIP file <a href="#">here</ a> .</ p> |
| − | <li>The pump should begin to rotate and the LEDs should light up. </li>
| + | |
| − | <li>Connect the Arduino controlling the 2-in-1 sensor to your PC. </li>
| + | |
| − | <li>Load the code for the 2-in-1 sensor and open the Serial Monitor to check that the sensor is collecting data. </li>
| + | |
| − | <li>After verifying that all the components are working to your satisfaction, close the shaking incubator door. </li>
| + | |
| − | <li>When the OD reaches your target levels, the LEDs in the fermentation chamber will turn off. The green LED in the 2-in-1 sensor will also turn off, and the red LED will turn on. The default OD in the code is 0.6.</li>
| + | |
| − | <li>When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are stressed, the LEDs in the fermentation chamber will turn on again.</li>
| + | |
| − | <li>When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are NOT stressed, the LEDs in the fermentation chamber will turn off again.</li>
| + | |
| − | <li>Steps 7-8 will repeat indefinitely, unless you power the system off.</li>
| + | |
| − | </ol>
| + | |
| − | <br>
| + | |
| − | <h4>Components</h4>
| + | |
| − | <br>
| + | |
| − | <p><i>Light Wait</i> comprises a peristaltic pump, a 2-in-1 OD and fluorescence sensor, and a fermentation chamber. Click on the picture of the component to be taken to its dedicated page!</p>
| + | |
| − | <br>
| + | |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Video goes here</b> : labelled picture of pump</figcaption>
| + | |
| − | </figure>
| + | |
| − | <br>
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| − | <button class="accordion"> TEST </button>
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| − | <h3><i>Abs<sub>600</sub></i></h3>
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| − | <ul style="list-style: none; margin: 0; padding: 1em; text-align:left; border-left: .5px solid black">
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| − | <li> Wavelength: 600nm </li>
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| − | <li> Read Speed: Normal </li>
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| − | <li> Delay: 100 msec </li>
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| − | <li> Excitation: 485 </li>
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| − | <li>Emission: 525</li>
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| − | <li>Optics: Top</li>
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| − | <li>Gain: 50</li>
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| − | <li>Light Source: Xenon Flash</li>
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| − | <li>Lamp Energy: High</li>
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| − | <li>Read Speed: Normal</li>
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| − | <li>Delay: 100 msec</li>
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| − | <li>Read Height: 7 mm</li>
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| − | <li> Wavelength: 600nm </li>
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| − | <li> Read Speed: Normal </li>
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| − | <li> Delay: 100 msec </li>
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| − | <li> Excitation: 485 </li>
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| − | <li>Emission: 525</li>
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| − | <li>Optics: Top</li>
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| − | <li>Gain: 50</li>
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| − | <li>Light Source: Xenon Flash</li>
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| − | <li>Lamp Energy: High</li>
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| − | <li>Read Speed: Normal</li>
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| − | <li>Delay: 100 msec</li>
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| − | <li>Read Height: 7 mm</li>
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| | <br> | | <br> |
| − | <h2> CONFIGURATIONS </h2> | + | <div style="text-align: left;"> |
| − | <br>
| + | <sup>[1]</sup>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876151/ |
| − | | + | </div> |
| − | <p> KITTY IPSUM dolor sit amet discovered siamesecalico peaceful her Gizmo peaceful boy rutrum caturday enim lived quis Mauris sit malesuada gf's saved fringilla enim turpis, at mi kitties ham. Venenatis belly cat et boy bat dang saved nulla other porta ipsum mi chilling cat spoon tellus.</p>
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| − | <figcaption><b>Video goes here</b> : blah blah 3</figcaption>
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| − | <img src="#" alt="Video">
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| − | <figcaption><b>Video goes here</b> : blah blah 3</figcaption>
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| − | </figure>
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| − | <br>
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| − | <br>
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| − | <figure class="figures">
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| − | <img src="#" alt="Video">
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| − | <figcaption><b>Video goes here</b> : blah blah 3</figcaption>
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| − | </figure>
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| − | <br>
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| − | <p> KITTY IPSUM dolor sit amet discovered siamesecalico peaceful her Gizmo peaceful boy rutrum caturday enim lived quis Mauris sit malesuada gf's saved fringilla enim turpis, at mi kitties ham. Venenatis belly cat et boy bat dang saved nulla other porta ipsum mi chilling cat spoon tellus.</p>
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| − | <br>
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| − | <h2>Bio-production</h2>
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| − | <p>It’s important to have automation in bioproduction especially in industrial level. We designed a small bioreactor system which incorporated optical density (OD) and fluorescence sensors to control the metabolic behaviours in E. coli. </p><br>
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| − | <h4>Automated Control through feedbacks</h4>
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| − | <p>IN progress. </p>
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| − | <h4>OD/F sensor</h4>
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| − | <p>IN progress.</p>
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| − | <h4>Pump</h4>
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| − | <p>IN progress.</p>
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| | </div></div> | | </div></div> |
