Difference between revisions of "Team:NUS Singapore-A/Hardware/Pump"

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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>
 
 
<br>
 
<br>
 
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<video src="https://static.igem.org/mediawiki/2018/8/8a/T--NUS_Singapore-A--Hardware_pump.mov" width:"640" height="480" controls Autoplay=autoplay>pump moving</video>
<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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<br>
 
<br>
  
<h2>Problem #1</h2>
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<h1>Peristaltic Pump</h1>
 +
<p>The peristaltic pump displaces fluid, such that bacterial culture is kept flowing through the <a href="#">cuvette</a>. This allows the <a href ="#">sensor</a> to take continuous measurements.</p>
 +
 
 +
<h2>Design - Improvement!</h2>
 +
<p>This peristaltic pump is an improvement on the design by the 2015 Aachen iGEM team. We chose to build on their design since their pump was also designed for continuous pumping in a bioreactor. Three modifications were made.</p>
 
<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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 +
<button class="accordion">IMPROVEMENT 1 - CIRCULAR ROTOR BASE</button>
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  <div class="panel">
 +
    <p>The first modification replaces their pentagonal spring rotor base with a circular rotor base (Figure 1).</p>
 +
 
 
<br>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/b/b8/T--NUS_Singapore-A--Hardware_pump_oldhub.png" style="max-width: 45%; display:inline-block;">
   <figcaption><b>Figure 1</b>. Scaling-up in optogenetics research - from the microplate to small-scale bioreactor</figcaption>
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  <img src="https://static.igem.org/mediawiki/2018/1/14/T--NUS_Singapore-A--Hardware_pump_newhub.png" style="max-width: 45%; display:inline-block;">
 +
   <figcaption><b>Figure 1</b>. Pentagonal spring rotor figure (left) against our rotor design (right)</figcaption>
 
</figure>
 
</figure>
 
<br>
 
<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>
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 +
<p>This was so that we could countersink flat-head bolts into the rotor structure, compared to having raised bolts such as pan-head bolts (Figure 2).</p>
 +
 
 
<br>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/3/32/T--NUS_Singapore-A--Hardware_Comparison_of_Screws_for_Rotor_HLR_White.png">
   <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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   <figcaption><b>Figure 2</b>. Aachen 2015’s rotor with a pan-head bolt installed (left) and our rotor with a flat-head bolt installed (right). The choice of bolt for the Aachen rotor was arbitrary and is for illustration purposes. Their wiki simply instructs one to use an M3 bolt there. Our flat-head bolt is flush with the bottom face of the rotor. The height of our rotor’s base was thickened slightly to accommodate the countersinking feature.</figcaption>
 
</figure>
 
</figure>
 
<br>
 
<br>
  
<h2>Problem #2</h2>
+
<p>When we recreated Aachen 2015’s pump, we discovered that no matter how carefully we mounted the rotor onto the shaft of the stepper motor, one screw head would always drag along the pump plates, implying that the rotor was tilted at an angle.This caused the pump to have insufficient torque to move fluid through the silicone tubes.</p>
<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>
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 +
<p>The screw attaching the rotor to the stepper motor shaft is a flat end screw, and the rotor should not be tilted at an angle. Troubleshooting further would yield diminishing marginal returns, as other possible sources of error such as inaccuracies in 3D-printing would not be within our power to resolve. Moreover, the angle was very slight and otherwise undetectable by visual inspection. An obvious solution would be to decrease the height of the central rotor column, and then shift the entire rotor assembly upwards, but this would not be ideal. As the load moves further along the shaft, the angular deflection of the shaft increases. While the increase in deflection would definitely be negligible in this case, such a solution would be bad design practice. Hence, we elected to countersink the bolts because this approach allows us to easily visually inspect the rotor to ensure clearance between it and the faceplate of the stepper motor.</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>
 
<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>
 
  
 +
<p>However, a flat-head bolt of equivalent size could not be countersunk directly into the Aachen design as there was too little material remaining to hold the bolt in place. We then simplified the rotor base, and thus produced what you see in Figure 2.</p>
  
<h3>PDF-LA!</h3>
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    <button class="accordion-closer">CLOSE</button>
<br>
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</div>
<h4>Function</h4>
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<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>
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<h4>Product Demonstration</h4>
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<button class="accordion">IMPROVEMENT 2 - ADDING MATERIAL FOR FASTENER</button>
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  <div class="panel">
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    <p>Our second modification was to add material for the fastening screw. A drawback of the 2015 team’s design was the weakness of the bolt and nut combination fastening the rotor to the stepper motor shaft (Figure 3).</p>
  
 
<br>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/b/b7/T--NUS_Singapore-A--Hardware_pump_fasteningscrew1.png">
  <figcaption><b>Figure 2</b>. gif in progress</figcaption>
+
   <figcaption><b>Figure 3</b>. The wall separating the fastening nut and the shaft was less than 0.5mm thick. When we attempted to 3D-print the Aachen 2015 rotor, our 3D printer was not precise enough to produce such a thickness, or rather, thinness. While the rotor was still functional in this state, we felt that this design had room for improvement. The type of fastening nut here used was arbitrary.</figcaption>
  <img src="#" alt="Video">
+
   <figcaption><b>Figure 1</b>. Showcase of PDF-LA!</figcaption>
+
 
</figure>
 
</figure>
 
<br>
 
<br>
  
<video src="#" width="300"> uploaded to drive </video>
+
  <p>We thickened the wall separating the fastening nut and the shaft by 2.75 mm, placing the nut 6mm away from the centre of the shaft (Figure 4). We also thickened the wall between the bolt head and the nut. This design can thus better withstand mechanical stresses, and is more durable. </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>
 
 
<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>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
+
   <img src="https://static.igem.org/mediawiki/2018/f/f7/T--NUS_Singapore-A--Hardware_pump_fasteningscrew2.png">
   <figcaption><b>Figure 2</b>GIF of plate, dish, flask going into each container</figcaption>
+
   <figcaption><b>Figure 4</b>. The circled parts show where we have added material around the fastening bolt for a sturdier structure. </figcaption>
 
</figure>
 
</figure>
 
<br>
 
<br>
<li>Connect the AC adapter to the Arduino and wall socket.</li>
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    <button class="accordion-closer">CLOSE</button>
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  </div>
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<button class="accordion">IMPROVEMENT 3 - REPOSITIONING INLET & OUTLET</button>
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  <div class="panel">
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    <p>The third modification we made was to reposition the inlet and outlet for the silicone tubing.</p>
 +
    <p>Aachen 2015’s design stacked several Plexiglass layers on the stepper motor’s faceplate, forming a housing to hold a silicone tube inside a circular path. We noticed that Aachen 2015’s silicone tubes had a smaller outer and inner diameter than the tubes we planned to use, as the working volume of their bioreactor is less than ours. After analyzing their design, we conjectured that we only needed to modify the thickness of the pumping layer (Figure 5) to accommodate our own silicone tubing.</p>
 +
 
 
<br>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/4/4e/T--NUS_Singapore-A--Hardware_pump_originallayer1.png">
   <figcaption><b>Figure 2</b>arduino, AC adapter picture, wall socket picture, arrows to indicate</figcaption>
+
   <figcaption><b>Figure 5</b>. Original shape of pumping layer.</figcaption>
 
</figure>
 
</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>
 
<br>
  
<h4>Possible Configurations</h4>
+
  <p>However, our silicone tube exerted forces on the walls of the pumping layer and caused the walls to deflect (Figure 6).</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>
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<br>
+
  
<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>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
+
   <img src="https://static.igem.org/mediawiki/2018/9/9b/T--NUS_Singapore-A--Hardware_pump_originallayer2.png" >
   <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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   <figcaption><b>Figure 6</b>. Diagram showing the direction of normal forces exerted by our silicone tubing on the pumping layer. The direction of deflection is the same as the force here.</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>
+
  <p>Because of this, the silicone tube could not be pinched closed (occluded), and the pump was unable to force the fluid to move through the tube.</p>
 +
  <p>We thus repositioned the inlet and outlet, combining them into one opening (Figure 7).</p>
 +
 
 
<br>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
+
   <img src="https://static.igem.org/mediawiki/2018/0/0b/T--NUS_Singapore-A--Hardware_pump_newlayer1.png">
   <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>
+
   <figcaption><b>Figure 7</b>. New shape of pumping layer.</figcaption>
 
</figure>
 
</figure>
 
<br>
 
<br>
  
 
+
  <p>Now, when the silicone tube attempts to regain its original, non-deformed shape, the lines of action of the forces it exerts at the opening will be much closer to the metal fasteners (Figure 8), causing a much smaller bending moment. Additionally, the force exerted by the silicone tube is now shared by 4 fasteners instead of 2 as the shape is now continuous.</p>
<p>Presenting, <i>PDF-LA!</i> Click <a href="#">here</a> for its dedicated page.</p>
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<br>
 
<br>
 
<figure class="figures">
 
<figure class="figures">
   <img src="#" alt="Video">
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   <img src="https://static.igem.org/mediawiki/2018/b/b6/T--NUS_Singapore-A--Hardware_pump_newlayer2.png">
   <figcaption><b>Figure 2</b><i>P-LA!</i> and <i>DF-LA!</i> side by side</figcaption>
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   <figcaption><b>Figure 8</b>. Diagram showing the direction of normal forces exerted by our silicone tubing on the new pumping layer.</figcaption>
 
</figure>
 
</figure>
<br>
 
<hr>
 
 
<br>
 
<br>
  
<h3>Light Wait</h3>
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<p>After this modification, the pumping layer no longer deflected visibly, and operation was smooth.</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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<br>
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<h4>Validation</h4>
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    <button class="accordion-closer">CLOSE</button>
<br>
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  </div>
<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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<br>
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<h2>Testing</h2>
<h4>Experimental Plans</h4>
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<p>We validated the functionality of this component and characterized it by plotting the mass flow rate as a function of RPM.</p>
<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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<button class="accordion">PROCEDURE</button>
 +
  <div class="panel">
 
<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>Place a length of silicone tubing in the peristaltic pump. Put one end into a water reservoir. Place another end into a clean, empty beaker. Put the empty beaker on an electronic balance.</li>
  <li>Fill and cover the fermentation chamber. </li>
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<li>Use the code to set the RPM.</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>Turn the peristaltic pump on.</li>
 
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<li>Wait approximately 5 seconds for the flow to stabilize. When the flow has stabilized, start the stopwatch while simultaneously taring the electronic balance.</li>
<br>
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<li>Read the mass off the display on the electronic balance every 10 seconds, for 5 minutes total.</li>
<figure class="figures">
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<li>After completing Step 4, stop the stopwatch and empty the beaker. Refill the water reservoir if necessary.</li>
  <img src="#" alt="Video">
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<li>Repeat Steps 3-5 twice more.</li>
  <figcaption><b>Figure __</b>. Illustration of how the pump, sensor, and fermentation chamber should be connected by silicone tubing.</figcaption>
+
<li>Represent the measurements with a scatter plot. Find the trendline. The gradient is the mass flow rate [g/s]. While kg is the correct SI unit, we find g to be more helpful here.</li>
</figure>
+
<li>Record mass flow rate and RPM.</li>
<br>
+
<li>Repeat steps 4-9 for different RPMs.</li>
 
+
<li>Plot a graph of flow rate against RPM. You may now use this graph to find out what RPM you should enter in the code for your desired mass flow rate.</li>
<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>
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<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>
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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>
+
 
</ol>
 
</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>
 
 
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    <button class="accordion-closer">CLOSE</button>
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  </div>
  
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<button class="accordion">RESULTS</button>
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    <div class="panel">
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  <p>Our pump was able to displace fluid consistently for different mass flow rates. Please visit <a href="#"><i>Results:Cell-Machine Interface</i></a> for the results of our characterization of this pump.</p>
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  <button class="accordion-closer">CLOSE</button>
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    </div>
  
  <button class="accordion"> TEST </button>
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<h2>Construction</h2>
    <div class="panel" style="line-height: 17em;">
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<p>Ever wanted a peristaltic pump of your own? Now you can have one, get started here!</p>
  
      <div class="row">
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<button class="accordion">BILL OF MATERIALS</button>
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     <div class="panel">
          <table>
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<p>All fasteners required are M3 size.</p>
            <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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            </tr>
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          </table>
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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">
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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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          </table>
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  <button class="accordion"> COMPONENTS </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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                </ul>
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                <h3><i>Fluorescence</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> 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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  <li>12V AC adapter x 1</li>
<h2> CONFIGURATIONS </h2>
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  <li>Stepper motor x 1</li>
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  <li>Motor drive x 1</li>
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  <li>ID=3 mm OD=7 mm radial ball bearing x 8</li>
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  <li>ID=3 mm OD=5 mm food grade heat-resistant silicone tube x 1</li>
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  <li>200mm x 200mm x 3mm clear acrylic sheet x 1</li>
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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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   <figcaption><b>Table 1</b>. List of components</figcaption>
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   <figcaption><b>Figure 10</b>. Rotor assembly for one countersunk bolt. Note that washer size as shown is inaccurate.</figcaption>
 
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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="https://static.igem.org/mediawiki/2018/0/04/T--NUS_Singapore-A--Hardware_Peristaltic_Pump.zip">here</a>.</p>
  
<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>IN progress.</p>
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Latest revision as of 23:54, 17 October 2018

CONNECT WITH US


Peristaltic Pump

The peristaltic pump displaces fluid, such that bacterial culture is kept flowing through the cuvette. This allows the sensor to take continuous measurements.

Design - Improvement!

This peristaltic pump is an improvement on the design by the 2015 Aachen iGEM team. We chose to build on their design since their pump was also designed for continuous pumping in a bioreactor. Three modifications were made.


The first modification replaces their pentagonal spring rotor base with a circular rotor base (Figure 1).


Figure 1. Pentagonal spring rotor figure (left) against our rotor design (right)

This was so that we could countersink flat-head bolts into the rotor structure, compared to having raised bolts such as pan-head bolts (Figure 2).


Figure 2. Aachen 2015’s rotor with a pan-head bolt installed (left) and our rotor with a flat-head bolt installed (right). The choice of bolt for the Aachen rotor was arbitrary and is for illustration purposes. Their wiki simply instructs one to use an M3 bolt there. Our flat-head bolt is flush with the bottom face of the rotor. The height of our rotor’s base was thickened slightly to accommodate the countersinking feature.

When we recreated Aachen 2015’s pump, we discovered that no matter how carefully we mounted the rotor onto the shaft of the stepper motor, one screw head would always drag along the pump plates, implying that the rotor was tilted at an angle.This caused the pump to have insufficient torque to move fluid through the silicone tubes.

The screw attaching the rotor to the stepper motor shaft is a flat end screw, and the rotor should not be tilted at an angle. Troubleshooting further would yield diminishing marginal returns, as other possible sources of error such as inaccuracies in 3D-printing would not be within our power to resolve. Moreover, the angle was very slight and otherwise undetectable by visual inspection. An obvious solution would be to decrease the height of the central rotor column, and then shift the entire rotor assembly upwards, but this would not be ideal. As the load moves further along the shaft, the angular deflection of the shaft increases. While the increase in deflection would definitely be negligible in this case, such a solution would be bad design practice. Hence, we elected to countersink the bolts because this approach allows us to easily visually inspect the rotor to ensure clearance between it and the faceplate of the stepper motor.


However, a flat-head bolt of equivalent size could not be countersunk directly into the Aachen design as there was too little material remaining to hold the bolt in place. We then simplified the rotor base, and thus produced what you see in Figure 2.

Our second modification was to add material for the fastening screw. A drawback of the 2015 team’s design was the weakness of the bolt and nut combination fastening the rotor to the stepper motor shaft (Figure 3).


Figure 3. The wall separating the fastening nut and the shaft was less than 0.5mm thick. When we attempted to 3D-print the Aachen 2015 rotor, our 3D printer was not precise enough to produce such a thickness, or rather, thinness. While the rotor was still functional in this state, we felt that this design had room for improvement. The type of fastening nut here used was arbitrary.

We thickened the wall separating the fastening nut and the shaft by 2.75 mm, placing the nut 6mm away from the centre of the shaft (Figure 4). We also thickened the wall between the bolt head and the nut. This design can thus better withstand mechanical stresses, and is more durable.


Figure 4. The circled parts show where we have added material around the fastening bolt for a sturdier structure.

The third modification we made was to reposition the inlet and outlet for the silicone tubing.

Aachen 2015’s design stacked several Plexiglass layers on the stepper motor’s faceplate, forming a housing to hold a silicone tube inside a circular path. We noticed that Aachen 2015’s silicone tubes had a smaller outer and inner diameter than the tubes we planned to use, as the working volume of their bioreactor is less than ours. After analyzing their design, we conjectured that we only needed to modify the thickness of the pumping layer (Figure 5) to accommodate our own silicone tubing.


Figure 5. Original shape of pumping layer.

However, our silicone tube exerted forces on the walls of the pumping layer and caused the walls to deflect (Figure 6).


Figure 6. Diagram showing the direction of normal forces exerted by our silicone tubing on the pumping layer. The direction of deflection is the same as the force here.

Because of this, the silicone tube could not be pinched closed (occluded), and the pump was unable to force the fluid to move through the tube.

We thus repositioned the inlet and outlet, combining them into one opening (Figure 7).


Figure 7. New shape of pumping layer.

Now, when the silicone tube attempts to regain its original, non-deformed shape, the lines of action of the forces it exerts at the opening will be much closer to the metal fasteners (Figure 8), causing a much smaller bending moment. Additionally, the force exerted by the silicone tube is now shared by 4 fasteners instead of 2 as the shape is now continuous.


Figure 8. Diagram showing the direction of normal forces exerted by our silicone tubing on the new pumping layer.

After this modification, the pumping layer no longer deflected visibly, and operation was smooth.

Testing

We validated the functionality of this component and characterized it by plotting the mass flow rate as a function of RPM.


  1. Place a length of silicone tubing in the peristaltic pump. Put one end into a water reservoir. Place another end into a clean, empty beaker. Put the empty beaker on an electronic balance.
  2. Use the code to set the RPM.
  3. Turn the peristaltic pump on.
  4. Wait approximately 5 seconds for the flow to stabilize. When the flow has stabilized, start the stopwatch while simultaneously taring the electronic balance.
  5. Read the mass off the display on the electronic balance every 10 seconds, for 5 minutes total.
  6. After completing Step 4, stop the stopwatch and empty the beaker. Refill the water reservoir if necessary.
  7. Repeat Steps 3-5 twice more.
  8. Represent the measurements with a scatter plot. Find the trendline. The gradient is the mass flow rate [g/s]. While kg is the correct SI unit, we find g to be more helpful here.
  9. Record mass flow rate and RPM.
  10. Repeat steps 4-9 for different RPMs.
  11. Plot a graph of flow rate against RPM. You may now use this graph to find out what RPM you should enter in the code for your desired mass flow rate.

Our pump was able to displace fluid consistently for different mass flow rates. Please visit Results:Cell-Machine Interface for the results of our characterization of this pump.

Construction

Ever wanted a peristaltic pump of your own? Now you can have one, get started here!

All fasteners required are M3 size.


  • 12V AC adapter x 1
  • Stepper motor x 1
  • Motor drive x 1
  • ID=3 mm OD=7 mm radial ball bearing x 8
  • ID=3 mm OD=5 mm food grade heat-resistant silicone tube x 1
  • 200mm x 200mm x 3mm clear acrylic sheet x 1


Figure 9. Pump assembly


Figure 10. Rotor assembly for one countersunk bolt. Note that washer size as shown is inaccurate.


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 here.