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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">
  
<h1>Introduction</h1>
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<h1>P-LA! and DF-LA!</h1>
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<p>Plate-Dish-Flask Light Apparatus (PDF-LA!) is a suite of three fully programmable devices compatible with standard laboratory equipment. It was used to characterize our optogenetic circuit in 12-well plates, petri dishes, and Erlenmeyer flasks.</p>
 
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<h2>Design - Innovation!</h2>
<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>
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<p>We designed PDF-LA! to help us characterize the behaviour of EL222. A research fellow sharing our lab, Dr Teh Ai Ying (Figure 1), was also interested in using our custom optogenetic tools, and so she served as our main source of user feedback throughout PDF-LA!’s design process.</p>
 
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<br>
  
<h2>Problem #1</h2>
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<div class="quote"> This was useful to me as I needed to scale up my research. </div>
 
<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>
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<figure class="figures2" style="max-width: 60%;">
<br>
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   <img src="https://static.igem.org/mediawiki/2018/6/60/T--NUS_Singapore-A--Hardware_pdfla_aiying.jpg" alt="Dr Teh and her satisfaction">
<figure class="figures">
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   <figcaption><b>Figure 1</b>. Dr Teh Ai Ying, holding DF-LA!.</figcaption>
   <img src="#" alt="Video">
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  <figcaption><b>Figure 1</b>. Scaling-up in optogenetics research - from the microplate to small-scale bioreactor</figcaption>
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</figure>
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<br>
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<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>
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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>Figure 2</b>. The components in Figure 1 (bottom right-hand corner) are still dwarfed by an industrial bioreactor.</figcaption>
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</figure>
 
</figure>
 
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<br>
 
<h2>Problem #2</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>
 
<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>
 
<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>
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<h3><i>P-LA!</i></h3>
<br>
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<p>The first design was a device for the 12-well plate, <i>P-LA!</i>. We designed <i>P-LA!</i> to improve on another design which had been loaned to us by a different university (Figure 2), and hence gain experience of creating custom tools for optogenetics research.</p>
<h4>Function</h4>
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<p><i>P-LA!</i> is better than the previous design because it can accommodate 12-well plates from different manufacturers, whereas the other design could only fit a specific 12-well plate model (Figure 2). P-LA! also uses significantly less material. Fun fact: despite its name, <i>P-LA!</i> was printed with ABS filament rather than PLA filament! All our 3D-printed designs use ABS filament as it was made readily available to us by our university, and its material properties were deemed sufficient for our purposes.</p>
<br>
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<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>
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<br>
 
<br>
 
<h4>Product Demonstration</h4>
 
  
 
<br>
 
<br>
<figure class="figures">
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<figure class="figures2" style="max-width: 95%;">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/e/e3/T--NUS_Singapore-A--Hardware_pdfla_12wellplate.png"style="display: inline-block; width: 50%;">
  <figcaption><b>Figure 2</b>. gif in progress</figcaption>
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    <img src="https://static.igem.org/mediawiki/2018/5/5a/T--NUS_Singapore-A--Hardware_Peristaltic_Pump.jpeg" style="display: inline-block; width: 50%;">
  <img src="#" alt="Video">
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   <figcaption><b>Figure 2</b>. Device for 12-Well Plate a.k.a. <i>P-LA!</i> (top), previous design which cannot accommodate other models of 12-well plate (bottom).</figcaption>
   <figcaption><b>Figure 1</b>. Showcase of PDF-LA!</figcaption>
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</figure>
 
</figure>
 
<br>
 
<br>
  
<video src="#" width="300"> uploaded to drive </video>
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<p>After optimizing our device for the 12-well plate, it was time to scale up and create devices for petri dishes and Erlenmeyer flasks. These two items of standard laboratory equipment were chosen based on feedback from research fellows in NUS. To provide users a more integrated solution, we produced a single device which can be used for both, <i>DF-LA!</i>. </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>
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<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>
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<br>
 
<br>
  
<h3>How it Works</h3>
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<h3><i>DF-LA!</i></h3>
<br>
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<p>As such a device had not been designed previously, there was no existing structure to improve or draw inspiration from, and our team started from scratch. This resulted in many design iterations (Figure 13).</p>
<h4>Operation</h4>
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<br>
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<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>
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<br>
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<ol>
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<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>
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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>Figure 2</b>GIF of plate, dish, flask going into each container</figcaption>
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</figure>
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<br>
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<li>Connect the AC adapter to the Arduino and wall socket.</li>
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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>Figure 2</b>arduino, AC adapter picture, wall socket picture, arrows to indicate</figcaption>
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</figure>
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<br>
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<li>Turn on the wall switch controlling the AC adapter. </li>
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<br>
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</ol>
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<br>
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<p>The devices should light up as shown in the product demonstration video above.</p>
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<br>
  
<h4>Possible Configurations</h4>
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<figure class="figures2" style="max-width: 95%;">
<br>
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  <img src="https://static.igem.org/mediawiki/2018/4/44/T--NUS_Singapore-A--Hardware_pdfla_iteration1.gif" style="display: inline-block; width: 24%;">
<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>
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  <img src="https://static.igem.org/mediawiki/2018/3/33/T--NUS_Singapore-A--Hardware_pdfla_iteration2.gif" style="display: inline-block; width: 24%;">
<br>
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  <img src="https://static.igem.org/mediawiki/2018/5/5f/T--NUS_Singapore-A--Hardware_pdfla_iteration3.gif" style="display: inline-block; width: 24%;">
 
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   <img src="https://static.igem.org/mediawiki/2018/c/ca/T--NUS_Singapore-A--Hardware_pdfla_iteration4.gif" style="display: inline-block; width: 24%;">
<p>Examples of the possible configurations can be found below.</p>
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   <figcaption><b>Figure 3</b>. The various design iterations, starting from left. This illustrates the design features that have persisted through testing and user feedback.</figcaption>
<br>
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<ul>
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<li>1 x D-LA!, bottom illu, bottom and top illu</li>
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<li>1 x F-LA!, bottom illu, bottom and top illu</li>
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<li>N x DF-LA!, bottom-bottom and bottom and top illu</li>
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</ul>
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<br>
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<h4>Components</h4>
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<br>
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<p><i>P-LA!</i> comprises a tech holder and a lighting plate</p>
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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>Figure 2</b>picture of tech holder, picture of lighting plate, GIF of collapsed assembled <i>P-LA!</i></figcaption>
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</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>
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<button class="accordion">First to Second Iteration</button>
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<div class="panel">
<figure class="figures">
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<p>The first iteration is for a single petri dish, which can be illuminated from top and bottom. Users wanted a system with the additional option to test two petri dishes at once, and the second iteration was conceived.</p>
  <img src="#" alt="Video">
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  <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>
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</figure>
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<br>
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<button class="accordion-closer">CLOSE</button>
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</div>
  
 
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<button class="accordion">Second to Third Iteration</button>
<p>Presenting, <i>PDF-LA!</i> Click <a href="#">here</a> for its dedicated page.</p>
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<div class="panel">
 
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<p>The second iteration was designed such that the components to illuminate a single petri dish from the bottom could be stacked with another set of the same components to create a structure that could expose the petri dish to light from the top and bottom. This symmetry is advantageous as the functionality of a single unit doubles, and was therefore retained and developed in subsequent design iterations.</p>
<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b><i>P-LA!</i> and <i>DF-LA!</i> side by side</figcaption>
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</figure>
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<br>
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<hr>
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<br>
 
<br>
  
<h3>Light Wait</h3>
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<p>After that, we attempted to design a separate device for the Erlenmeyer flask, but realized that it would be much simpler and more elegant to adapt the second iteration to also accommodate the flask. The third iteration thus has an extra ring in between the devices for the petri dish. This acts as a holder for the flask. Also, the “teeth and slots” feature used to join the components together was moved from the center of the walls of the cylinder to the outer edge of the cylinder. We intended to shake the flask, and were thus concerned that the walls of the slot and the teeth would be too thin and might break due to shear stresses. We thus modified this feature for the third iteration. </p>
<br>
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<h4>Function</h4>
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<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>
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<br>
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<h4>Product Demonstration</h4>
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<br>
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<video src="#" width:"300">Bioreactor Backup Video</video>
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<br>
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<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>
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<button class="accordion-closer">CLOSE</button>
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</div>
  
<h4>Validation</h4>
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<button class="accordion">Third Iteration to Fourth Iteration</button>
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<div class="panel">
<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>
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<p>Major changes from the third iteration to the fourth iteration are the increase in the height of the flask adapter so that the flask could also be illuminated from both top and bottom, the increase in the number of LEDs available to illuminate the flask or dish, and modifying the shape of the electronics container so that the whole structure could be cylindrical. The reason for the last change was fivefold.</p>
<br>
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<h4>Experimental Plans</h4>
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<br>
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<h4>Experimental Results</h4>
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<br>
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<h4>How it Works</h4>
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<br>
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<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>
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<br>
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<h4>Operation</h4>
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<br>
 
<br>
 
<ol>
 
<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>
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<li>To reduce the material expended during 3D printing </li>
  <li>Fill and cover the fermentation chamber. </li>
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<li>For the device to occupy a smaller area </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>
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<li>For the user’s comfort during transport as the inconsistency in shape in previous iterations made the structure unwieldy to carry</li>
 
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<li>To make the design aesthetically pleasing</li>
<br>
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<li>Last but definitely not least, to make the design infinitely more modular as users can now continuously stack combinations of <i>D-LA!</i>, the petri dish module, and <i>F-LA!</i>, the Erlenmeyer flask module, into a single column. As the components are lightweight, structural failure is not a concern, and the number of units a user can stack is only limited by the microcontroller. Many petri dishes and flasks can be illuminated simultaneously while still occupying the same area as a single testing unit. The various possible configurations of our final iteration of <i>DF-LA!</i> can be seen on our <a href="https://2018.igem.org/Team:NUS_Singapore-A/Hardware">Hardware</a> page.</li>
<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure __</b>. Illustration of how the pump, sensor, and fermentation chamber should be connected by silicone tubing.</figcaption>
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</figure>
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<br>
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<li>Turn on the AC adapters for the pump and the LEDs in the fermentation chamber.</li>
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<li>The pump should begin to rotate and the LEDs should light up. </li>
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<li>Connect the Arduino controlling the 2-in-1 sensor to your PC. </li>
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<li>Load the code for the 2-in-1 sensor and open the Serial Monitor to check that the sensor is collecting data. </li>
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<li>After verifying that all the components are working to your satisfaction, close the shaking incubator door. </li>
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<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>
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<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>
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<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>
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<li>Steps 7-8 will repeat indefinitely, unless you power the system off.</li>
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</ol>
 
</ol>
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<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>
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<p>Dr Teh reported that the final design iteration successfully fulfilled her needs.</p>
 
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<br>
<figure class="figures">
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<button class="accordion-closer">CLOSE</button>
  <img src="#" alt="Video">
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  <figcaption><b>Video goes here</b> : labelled picture of pump</figcaption>
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</figure>
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</div>
 
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  <button class="accordion"> TEST </button>
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    <div class="panel" style="line-height: 17em;">
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      <div class="row">
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        <div class="column left">
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          <table>
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            <tr>
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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>
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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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          </table>
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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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            </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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      <div class="row">
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          <table>
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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>
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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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          <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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            </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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<hr>
 
<hr>
 
<br>
 
<br>
<h2> CONFIGURATIONS </h2>
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<h2>Testing</h2>
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<p>
 +
We validated the functionality of this component by using it to characterize the behaviour of the EL222 protein in blue light-repressible/inducible systems. Please see our <a href="https://2018.igem.org/Team:NUS_Singapore-A/Results">Results</a> page for an example of the results you can obtain if you use <i>PDF-LA!</i> to characterize your own optogenetic circuit.
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</p>
 
<br>
 
<br>
 
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<h2>Construction</h2>
<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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<p>
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You know you want <i>PDF-LA!</i>, and you’ve already chosen the googly eyes you’re going to stick on it. Here’s what you need to make your own.
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</p>
 
<br>
 
<br>
<figure class="figures">
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<h3>Bill of Materials</h3>
  <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>
 
<br>
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<p>As we were using PDF-LA! to characterize EL222, we bought 472 nm LEDs. You may substitute our LEDs with your own. The number of LEDs here are sufficient to build one set of P-LA! and one set of DF-LA!. Googly eyes have not been included. </p>
 
<br>
 
<br>
<figure class="figures">
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<ul>
   <img src="#" alt="Video">
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   <li>Arduino Uno x 3</li>
   <figcaption><b>Video goes here</b> : blah blah 3</figcaption>
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   <li>472 nm LED x 44</li>
</figure>
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  <li>LED driver TLC5940 x 3</li>
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  <li>0.1 uF ceramic capacitor x 3</li>
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  <li>4.7 uF electrolytic capacitor x 3</li>
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  <li>28-pin IC socket x 3</li>
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  <li>9V AC adapter x 3</li>
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</ul>
 
<br>
 
<br>
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<h3>Structural Assembly</h3>
 
<br>
 
<br>
<figure class="figures">
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<figure class="figures2" style="max-width: 70%;">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/6/6e/T--NUS_Singapore-A--Hardware_pdfla_12well_reverse.gif" alt="12-well" style="display: inline-block; width:60%;">
   <figcaption><b>Video goes here</b> : blah blah 3</figcaption>
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  <img src="https://static.igem.org/mediawiki/2018/d/dd/T--NUS_Singapore-A--Hardware_pdfla_iteration4_reverse.gif" alt="PDF-LA!" style="display: inline-block; width:30%;">
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   <figcaption><b>Figure 4</b>. Assemblies for P-LA! (left) and DF-LA! (right)</figcaption>
 
</figure>
 
</figure>
 
<br>
 
<br>
<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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<p>Files for lasercutting are available in DXF format, while files for 3D printing are available in STL format. You may download them all as a ZIP file <a href="#">here</a>.
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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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<p id="a_ref">Users reported satisfaction with the final design iteration. Please visit our <a href="https://2018.igem.org/Team:NUS_Singapore-A/Hardware"  id="b_ref">Hardware</a> page for more on how we conducted user testing and learned from user feedback.</p>
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<h4>OD/F sensor</h4>
 
<p>IN progress.</p>
 
  
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Latest revision as of 03:56, 18 October 2018

CONNECT WITH US

P-LA! and DF-LA!

Plate-Dish-Flask Light Apparatus (PDF-LA!) is a suite of three fully programmable devices compatible with standard laboratory equipment. It was used to characterize our optogenetic circuit in 12-well plates, petri dishes, and Erlenmeyer flasks.


Design - Innovation!

We designed PDF-LA! to help us characterize the behaviour of EL222. A research fellow sharing our lab, Dr Teh Ai Ying (Figure 1), was also interested in using our custom optogenetic tools, and so she served as our main source of user feedback throughout PDF-LA!’s design process.


This was useful to me as I needed to scale up my research.

Dr Teh and her satisfaction
Figure 1. Dr Teh Ai Ying, holding DF-LA!.

P-LA!

The first design was a device for the 12-well plate, P-LA!. We designed P-LA! to improve on another design which had been loaned to us by a different university (Figure 2), and hence gain experience of creating custom tools for optogenetics research.

P-LA! is better than the previous design because it can accommodate 12-well plates from different manufacturers, whereas the other design could only fit a specific 12-well plate model (Figure 2). P-LA! also uses significantly less material. Fun fact: despite its name, P-LA! was printed with ABS filament rather than PLA filament! All our 3D-printed designs use ABS filament as it was made readily available to us by our university, and its material properties were deemed sufficient for our purposes.



Figure 2. Device for 12-Well Plate a.k.a. P-LA! (top), previous design which cannot accommodate other models of 12-well plate (bottom).

After optimizing our device for the 12-well plate, it was time to scale up and create devices for petri dishes and Erlenmeyer flasks. These two items of standard laboratory equipment were chosen based on feedback from research fellows in NUS. To provide users a more integrated solution, we produced a single device which can be used for both, DF-LA!.


DF-LA!

As such a device had not been designed previously, there was no existing structure to improve or draw inspiration from, and our team started from scratch. This resulted in many design iterations (Figure 13).


Figure 3. The various design iterations, starting from left. This illustrates the design features that have persisted through testing and user feedback.

The first iteration is for a single petri dish, which can be illuminated from top and bottom. Users wanted a system with the additional option to test two petri dishes at once, and the second iteration was conceived.


The second iteration was designed such that the components to illuminate a single petri dish from the bottom could be stacked with another set of the same components to create a structure that could expose the petri dish to light from the top and bottom. This symmetry is advantageous as the functionality of a single unit doubles, and was therefore retained and developed in subsequent design iterations.


After that, we attempted to design a separate device for the Erlenmeyer flask, but realized that it would be much simpler and more elegant to adapt the second iteration to also accommodate the flask. The third iteration thus has an extra ring in between the devices for the petri dish. This acts as a holder for the flask. Also, the “teeth and slots” feature used to join the components together was moved from the center of the walls of the cylinder to the outer edge of the cylinder. We intended to shake the flask, and were thus concerned that the walls of the slot and the teeth would be too thin and might break due to shear stresses. We thus modified this feature for the third iteration.


Major changes from the third iteration to the fourth iteration are the increase in the height of the flask adapter so that the flask could also be illuminated from both top and bottom, the increase in the number of LEDs available to illuminate the flask or dish, and modifying the shape of the electronics container so that the whole structure could be cylindrical. The reason for the last change was fivefold.


  1. To reduce the material expended during 3D printing
  2. For the device to occupy a smaller area
  3. For the user’s comfort during transport as the inconsistency in shape in previous iterations made the structure unwieldy to carry
  4. To make the design aesthetically pleasing
  5. Last but definitely not least, to make the design infinitely more modular as users can now continuously stack combinations of D-LA!, the petri dish module, and F-LA!, the Erlenmeyer flask module, into a single column. As the components are lightweight, structural failure is not a concern, and the number of units a user can stack is only limited by the microcontroller. Many petri dishes and flasks can be illuminated simultaneously while still occupying the same area as a single testing unit. The various possible configurations of our final iteration of DF-LA! can be seen on our Hardware page.

Dr Teh reported that the final design iteration successfully fulfilled her needs.






Testing

We validated the functionality of this component by using it to characterize the behaviour of the EL222 protein in blue light-repressible/inducible systems. Please see our Results page for an example of the results you can obtain if you use PDF-LA! to characterize your own optogenetic circuit.


Construction

You know you want PDF-LA!, and you’ve already chosen the googly eyes you’re going to stick on it. Here’s what you need to make your own.


Bill of Materials


As we were using PDF-LA! to characterize EL222, we bought 472 nm LEDs. You may substitute our LEDs with your own. The number of LEDs here are sufficient to build one set of P-LA! and one set of DF-LA!. Googly eyes have not been included.


  • Arduino Uno x 3
  • 472 nm LED x 44
  • LED driver TLC5940 x 3
  • 0.1 uF ceramic capacitor x 3
  • 4.7 uF electrolytic capacitor x 3
  • 28-pin IC socket x 3
  • 9V AC adapter x 3

Structural Assembly


12-well PDF-LA!
Figure 4. Assemblies for P-LA! (left) and DF-LA! (right)