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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"> |
| | <br> | | <br> |
| − | <figure class="figures"> | + | |
| | + | <h1>2-in-1 OD and Fluorescence Sensor (INNOVATION)</h1> |
| | <img src="https://static.igem.org/mediawiki/2018/3/35/T--NUS_Singapore-A--The_Real_Sensor.png"> | | <img src="https://static.igem.org/mediawiki/2018/3/35/T--NUS_Singapore-A--The_Real_Sensor.png"> |
| − | </figure>
| |
| − | <h1>Introduction</h1>
| |
| | <br> | | <br> |
| | | | |
| − | <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> | + | <p>The 2-in-1 optical density (OD) and fluorescence sensor takes continuous OD and fluorescence readings of samples of the bacterial culture. These readings are feedback that will be used to control the activation of the LEDs in the fermentation chamber, and thus regulate metabolic flux.</p> |
| | <br> | | <br> |
| | | | |
| − | <h2>Problem #1</h2> | + | <h2>Design</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 integrated sensor consists of two structures - a cuvette through which bacterial culture flows, and a casing to contain the 2-in-1 sensing system and the cuvette. We capitalized on the fact that the emission wavelength of RFP (See Design: Stress Reporter), 600 nm, is the same as the wavelength at which the OD of a sample is conventionally measured.</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> | + | <h3>Structure - Cuvette</h3> |
| − | <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>The irregular shape of commercial cuvettes made an analytical understanding of the light paths passing through it difficult (Figure 1). Thus it was challenging to design a sensing system around it. Since a commercial cuvette is small, modifying it directly is also difficult.</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>PDF-LA!</h3>
| |
| − | <br>
| |
| − | <h4>Function</h4>
| |
| − | <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>
| |
| − | <br>
| |
| − |
| |
| − | <h4>Product Demonstration</h4>
| |
| − |
| |
| − | <br>
| |
| | <figure class="figures"> | | <figure class="figures"> |
| − | <img src="#" alt=" Video"> | + | <img src="#" alt=" Fig 1"> |
| − | <figcaption><b>Figure 2</b>. gif in progress</figcaption>
| + | <figcaption><b>Figure 1</b>. An example of a commercial cuvette. As the end of the cuvette is tapered and its surface has many extrusions, the surface area we could readily use for our measurements decreased. |
| − | <img src="#" alt="Video">
| + | </figcaption> |
| − | <figcaption><b>Figure 1</b>. Showcase of PDF-LA!</figcaption>
| + | |
| | </figure> | | </figure> |
| − | <br>
| |
| | | | |
| − | <video src="#" width="300"> uploaded to drive </video> | + | <p>We thus decided to design our own cuvette (Figure 2) using 1.5 mm thick acrylic sheets. Apart from allowing us to ensure that the walls are uniform, it also allowed us flexibility in the placement of the acrylic tubes channeling media through the cuvette. </p> |
| − | <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> | | <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"> | | <figure class="figures"> |
| − | <img src="#" alt=" Video"> | + | <img src="#" alt=" Fig 2"> |
| − | <figcaption><b>Figure 2</b>GIF of plate, dish, flask going into each container</figcaption>
| + | <figcaption><b>Figure 1</b>. All our cuvette’s surfaces are flat. |
| | + | </figcaption> |
| | </figure> | | </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>
| |
| − | <p>The devices should light up as shown in the product demonstration video above.</p>
| |
| − | <br>
| |
| | | | |
| − | <h4>Possible Configurations</h4>
| + | <p>Our cuvette was designed to have as few unique parts as possible. This was to facilitate easy assembly, which is an important design consideration since the cuvette is small. We would need to be able to quickly fabricate many cuvettes for testing. </p> |
| − | <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> | + | |
| | <br> | | <br> |
| | + | <p>This design choice proved useful when we were troubleshooting the cause of our cuvette’s leakiness. Acrylic glue could not form seamless bonds. Even if it appeared so from visual inspection, leaks sprung once we tested the cuvettes by pumping water through them at high pressure. After several rounds of trial and error with other adhesives, we discovered that Acrifix 1R 0912 UV adhesive formed a watertight seal and is clear when cured. We used it in our final design iteration for the cuvette.</p> |
| | | | |
| − | <p>Examples of the possible configurations can be found below.</p>
| |
| − | <br>
| |
| − | <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>
| |
| − | <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>Structure - Casing</h3> |
| | <br> | | <br> |
| − | <figure class="figures"> | + | <p>The casing houses the sensing system, which comprises a TSL235R light-to-frequency converter, a 600 nm LED, a 535 nm LED, and a LEE filter with a peak transmission of 600 nm. (Figure 3). The 600 nm LED is in the slot opposite the TSL235R, and is used to measure optical density (OD). The 535 nm is in the other LED slot, and is used to measure fluorescence. The LEE filter needs to be cut to an appropriate size and attached over the TSL235R. We used masking tape to secure the filter. </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> | | <br> |
| | | | |
| − | | + | <h3>Feedback Control</h3> |
| − | <p>Presenting, <i>PDF-LA!</i> Click <a href="#">here</a> for its dedicated page.</p> | + | <p> Will put flow chart</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>
| |
| − | <hr>
| |
| | <br> | | <br> |
| | | | |
| − | <h3>Light Wait</h3> | + | <h2>Testing</h2> |
| − | <br>
| + | <p>We validated the functionality of this component and characterized it by plotting graphs of sensor output frequencies against RFP readings measured using the NanoDrop. Our feedback control system does not require the simultaneous measurement of OD and fluorescence. Moreover, the different LEDs involved would not both be activated at the same time, and hence would not interfere with each other. We were thus able to calibrate OD and fluorescence separately. </p> |
| − | <h4>Function</h4>
| + | |
| − | <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>
| + | |
| | | | |
| − | <h4>Validation</h4> | + | <h3>Procedure</h3> |
| − | <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>
| + | |
| − | <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>
| + | |
| | | | |
| − | <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>
| |
| | | | |
| − | <br> | + | <h3>Results</h3> |
| − | <figure class="figures">
| + | |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Figure __</b>. Illustration of how the pump, sensor, and fermentation chamber should be connected by silicone tubing.</figcaption>
| + | |
| − | </figure>
| + | |
| − | <br>
| + | |
| | | | |
| − | <li>Turn on the AC adapters for the pump and the LEDs in the fermentation chamber.</li>
| |
| − | <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> | | <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> | + | <h2>Construction</h2> |
| | <br> | | <br> |
| − | <figure class="figures"> | + | <p>It’s beautiful. It’s obscenely integrated. It’s something you want right now. So why don’t you make it? We’ll help you!</p> |
| − | <img src="#" alt="Video">
| + | |
| − | <figcaption><b>Video goes here</b> : labelled picture of pump</figcaption>
| + | |
| − | </figure> | + | |
| | <br> | | <br> |
| | | | |
| | + | <h3>Bill of Materials</h3> |
| | | | |
| − | <button class="accordion"> TEST </button>
| + | <h3>Structural Assembly</h3> |
| − | <div class="panel" style="line-height: 17em;">
| + | |
| | | | |
| − | <div class="row">
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| − | <div class="column left">
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| − | <table>
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| − | <td style="padding:0;">
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| − | <h3><i>Abs<sub>600</sub></i></h3>
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| − | </td>
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| − | <td>
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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>
| |
| − | <li> Read Speed: Normal </li>
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| − | <li> Delay: 100 msec </li>
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | </div>
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| − | <div class="column right">
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| − | <table>
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| − | <tr>
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| − | <td style="padding:0;">
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| − | <h3><i>Fluorescence</i></h3>
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| − | </td>
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| − | <td>
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| − | <ul style="list-style: none; margin: 0; padding: 1em; text-align:left; border-left: .5px solid black">
| |
| − | <li> Excitation: 485 </li>
| |
| − | <li>Emission: 525</li>
| |
| − | <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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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <button class="accordion"> COMPONENTS </button>
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| − | <div class="panel" style="line-height: 17em;">
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| − |
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| − | <div class="row">
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| − | <div class="column left">
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| − | <td style="padding:0;">
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| − | <h3><i>Abs<sub>600</sub></i></h3>
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| − | <td>
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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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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | <div class="column right">
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| − | <table>
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| − | <td style="padding:0;">
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| − | <h3><i>Fluorescence</i></h3>
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| − | </td>
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| − | <td>
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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> 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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| − | </ul>
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| − | </td>
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| − | </table>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <br>
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| − | <hr>
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| − | <br>
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| − | <h2> CONFIGURATIONS </h2>
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| − | <br>
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| − |
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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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| | <figure class="figures"> | | <figure class="figures"> |
| | <img src="#" alt="Video"> | | <img src="#" alt="Video"> |
| − | <figcaption><b>Video goes here</b> : blah blah 3</figcaption> | + | <figcaption><b>Figure 2</b>Cuvette assembly</figcaption> |
| | </figure> | | </figure> |
| | <br> | | <br> |
| − | <br>
| + | |
| | <figure class="figures"> | | <figure class="figures"> |
| | <img src="#" alt="Video"> | | <img src="#" alt="Video"> |
| − | <figcaption><b>Video goes here</b> : blah blah 3</figcaption> | + | <figcaption><b>Figure 2</b>Sensor assembly</figcaption> |
| | </figure> | | </figure> |
| − | <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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| − | <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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| − | <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> |
