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

 
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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>
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<img src="https://static.igem.org/mediawiki/2018/3/35/T--NUS_Singapore-A--The_Real_Sensor.png">
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<h1>2-in-1 OD and Fluorescence Sensor (INNOVATION)</h1><br>
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<br>
 
<br>
  
 +
<h1>2-in-1 OD and Fluorescence Sensor</h1>
 
<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>
 
<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>
  
−
<img src="https://static.igem.org/mediawiki/2018/3/35/T--NUS_Singapore-A--The_Real_Sensor.png" style="width: 70%;">
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<h2>Design - Innovation!</h2>
 +
<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 <a href="https://2018.igem.org/Team:NUS_Singapore-A/Design#LUT">Design: Stress Reporter</a>), 600 nm, is the same as the wavelength at which the OD of a sample is conventionally measured, and aimed to design an integrated OD and fluorescence sensor.</p>
  
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<h2>Design</h2>
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<button class="accordion">STRUCTURE - CUVETTE</button>
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<br>
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  <div class="panel">
−
<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>
+
<p>The irregular shape of common commercial cuvettes made an analytical understanding of the light paths passing through it difficult. Thus it was challenging to design a sensing system around it. While there are straight-sided commercial cuvettes, they are so small that modifying them directly is not feasible, especially since we wished to modify the cuvette such that bacterial culture could flow through it continuously.</p>
 +
 
 +
<p>We thus decided to design our own cuvette (Figure 1) using 1.5 mm thick acrylic sheets. Apart from allowing us to ensure that the walls are flat, it also allowed us flexibility in the placement of the acrylic tubes channeling media through the cuvette. </p>
  
−
<h3>Structure - Cuvette</h3>
 
 
<br>
 
<br>
−
<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>
 
−
 
 
<figure class="figures">
 
<figure class="figures">
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     <img src="#" alt="Fig 1">
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     <img src="https://static.igem.org/mediawiki/2018/1/12/T--NUS_Singapore-A--Hardware_Cuvette_Collapsed_HLR_White.png" alt="Fig 1">
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     <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.
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     <figcaption><b>Figure 1</b>.Our DIY acrylic cuvette.
 
     </figcaption>
 
     </figcaption>
 
</figure>
 
</figure>
 +
<br>
  
−
<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>
+
<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>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>
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 +
<button class="accordion-closer">CLOSE</button>
 +
</div>
 +
 +
<button class="accordion">STRUCTURE - CASING</button>
 +
  <div class="panel">
 +
<p>The 3D-printed 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>
 +
 +
<br>
 
<figure class="figures">
 
<figure class="figures">
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     <img src="#" alt="Fig 2">
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     <img src="https://static.igem.org/mediawiki/2018/6/6a/T--NUS_Singapore-A--Hardware_Sensor_Bottom_Casing_Detailed.png" alt="Fig 2">
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     <figcaption><b>Figure 1</b>. All our cuvette’s surfaces are flat.
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     <figcaption><b>Figure 2</b>. Bottom casing of our sensor, with diagram showing where each component should be.
 
     </figcaption>
 
     </figcaption>
 
</figure>
 
</figure>
−
 
−
<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>
 
<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>
 
  
 +
<button class="accordion-closer">CLOSE</button>
 +
</div>
  
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<h3>Structure - Casing</h3>
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<button class="accordion">FEEDBACK CONTROL</button>
−
<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>
+
  <div class="panel">
−
 
+
    <p>A block diagram of our feedback control system is shown below (Figure 3).</p>
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<h3>Feedback Control</h3>
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    <br>
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<p> Will put flow chart</p>
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    <figure class="figures">
−
 
+
        <img src="https://static.igem.org/mediawiki/2018/f/f8/T--NUS_Singapore-A--Hardware_Feedback_Control.png">
 +
        <figcaption><b>Figure 3</b>. Blue light is turned off once the bacterial culture’s OD = 0.6. As the cells synthesize luteolin in the bioreactor, RFP expression levels increase as cell stress increases. RFP expression levels will be measured by the fluorescence sensor. When RFP expression levels reach 900 (as measured using a microplate reader (H1, Biotek, USA)), the blue light is turned on. Once the RFP expression levels are acceptable, the blue light is turned off. This cycle repeats.
 +
        </figcaption>
 +
    </figure>
 +
    <br>
 +
    <button class="accordion-closer">CLOSE</button>
 +
  </div>
 
<br>
 
<br>
  
 
<h2>Testing</h2>
 
<h2>Testing</h2>
−
<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>
+
<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 in the real system, and hence would not interfere with each other. We were thus able to calibrate OD and fluorescence separately.</p>
  
−
<h3>Procedure</h3>
+
<button class="accordion">PROCEDURE - OD SENSOR CALIBRATION</button>
 +
  <div class="panel">
 +
    <br>
 +
    <ol>
 +
    <li>Prepare 20 ml of E.coli cells harbouring Brep-RFP-YbaQ at OD = 3. These cells should have been previously been continuously exposed to blue light to repress RFP expression. </li>
 +
    <li>Dilute the cell medium to get 12 different samples with ODs ranging from 0.1 to 3. Label them from 1 - 12.</li>
 +
    <li>Measure the actual OD of sample 1 using the NanoDrop. </li>
 +
    <li>Transfer sample 1 into the cuvette with a syringe. Place the cuvette into the casing and close it. </li>
 +
    <li>Using PLX-DAQ, obtain approximately 10 readings of TSL235R’s output frequency when the 600 nm LED is lit.</li>
 +
    <li>Repeat steps 3-5 for the rest of the samples.</li>
 +
    <li>Plot a graph of output frequency against OD readings from the NanoDrop.</li>
 +
    </ol>
 +
    <br>
 +
    <button class="accordion-closer">CLOSE</button>
 +
  </div>
  
 +
<button class="accordion">PROCEDURE - FLUORESCENCE SENSOR CALIBRATION</button>
 +
  <div class="panel">
 +
    <br>
 +
    <ol>
 +
    <li>Prepare 20 ml of E.coli cells harbouring Brep-RFP-YbaQ at RFP reading = 7100. </li>
 +
    <li>Dilute the cell medium to get 12 different samples with RFP readings ranging from 100 to 7100. Label them from 1-12.</li>
 +
    <li>Measure the actual RFP reading of sample 1 using the NanoDrop. </li>
 +
    <li>Transfer sample 1 into the cuvette with a syringe. Place the cuvette into the casing and close it. </li>
 +
    <li>Using PLX-DAQ, obtain approximately 10 readings of TSL235R’s output frequency when the 535 nm LED is lit.This LED excites the RFP, causing it to fluoresce red with a wavelength of 600nm.</li>
 +
    <li>Repeat steps 3-5 for the rest of the samples.</li>
 +
    <li>Plot a graph of output frequency against RFP readings from the NanoDrop.</li>
 +
    </ol>
 +
    <br>
 +
    <button class="accordion-closer">CLOSE</button>
 +
  </div>
  
−
<h3>Results</h3>
+
<button class="accordion">RESULTS</button>
 +
  <div class="panel">
 +
<p>By following our experimental procedure, we were able to plot calibration curves for OD and fluorescence sensing. Please visit <a href="https://2018.igem.org/Team:NUS_Singapore-A/Results#BLS">Results:Cell-Machine Interface</a> for the results of our characterization of this sensor.</p>
 +
  <button class="accordion-closer">CLOSE</button>
 +
  </div>
  
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<br>
 
 
<h2>Construction</h2>
 
<h2>Construction</h2>
 
<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>
 
<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>
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<br>
 
  
−
<h3>Bill of Materials</h3>
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<button class="accordion">BILL OF MATERIALS</button>
 +
    <div class="panel">
 +
      <br>
 
<ul>
 
<ul>
−
   <li>Arduino Uno x 1</li>
+
   <li>Arduino Uno x 1</li>
 
   <li>600 nm LED x 1</li>
 
   <li>600 nm LED x 1</li>
 
   <li>535 nm LED x 1</li>
 
   <li>535 nm LED x 1</li>
 
   <li>Light-to-frequency converter TSL235R x 1</li>
 
   <li>Light-to-frequency converter TSL235R x 1</li>
 
   <li>LEE filter (peak transmission 600 nm)</li>
 
   <li>LEE filter (peak transmission 600 nm)</li>
−
   <li>ID = 3 mm acrylic tubes x 1 m</li>
+
   <li>OD=6 mm ID=3 mm acrylic tubes x 1 m</li>
 
   <li>Acrifix 1R 0912 UV Adhesive</li>
 
   <li>Acrifix 1R 0912 UV Adhesive</li>
 
</ul>
 
</ul>
−
<h3>Structural Assembly</h3>
+
<br>
 +
<button class="accordion-closer">CLOSE</button>
 +
  </div>
  
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<figure class="figures">
+
<button class="accordion">STRUCTURAL ASSEMBLY</button>
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  <img src="#" alt="Video">
+
    <div class="panel">
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  <figcaption><b>Figure 2</b>Cuvette assembly</figcaption>
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      <br>
−
</figure>
+
      <figure class="figures">
 +
        <img src="https://static.igem.org/mediawiki/2018/9/93/T--NUS_Singapore-A--Hardware_Cuvette_Assembly_HLR_White.gif" alt="Video">
 +
        <figcaption><b>Figure 4</b>. Cuvette assembly</figcaption>
 +
      </figure>
 +
      <br>
 +
      <br>
 +
      <figure class="figures">
 +
        <img src="https://static.igem.org/mediawiki/2018/f/f5/T--NUS_Singapore-A--Hardware_Integrated_Sensor_Assembly.gif" alt="Video">
 +
        <figcaption><b>Figure 5</b>. Sensor Assembly</figcaption>
 +
      </figure>
 +
      <br>
 +
      <button class="accordion-closer">CLOSE</button>
 +
    </div>
 +
<br>
 +
<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/2018/2/26/T--NUS_Singapore-A--Hardware_Sensor.zip">here</a>.
 +
<br>
 
<br>
 
<br>
  
−
<figure class="figures">
+
</div>
−
  <img src="#" alt="Video">
+
</div>
−
  <figcaption><b>Figure 2</b>Sensor assembly</figcaption>
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</figure>
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Latest revision as of 23:47, 17 October 2018

CONNECT WITH US


2-in-1 OD and Fluorescence Sensor

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.

Design - Innovation!

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, and aimed to design an integrated OD and fluorescence sensor.

The irregular shape of common commercial cuvettes made an analytical understanding of the light paths passing through it difficult. Thus it was challenging to design a sensing system around it. While there are straight-sided commercial cuvettes, they are so small that modifying them directly is not feasible, especially since we wished to modify the cuvette such that bacterial culture could flow through it continuously.

We thus decided to design our own cuvette (Figure 1) using 1.5 mm thick acrylic sheets. Apart from allowing us to ensure that the walls are flat, it also allowed us flexibility in the placement of the acrylic tubes channeling media through the cuvette.


Fig 1
Figure 1.Our DIY acrylic cuvette.

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.

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.

The 3D-printed 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.


Fig 2
Figure 2. Bottom casing of our sensor, with diagram showing where each component should be.

A block diagram of our feedback control system is shown below (Figure 3).


Figure 3. Blue light is turned off once the bacterial culture’s OD = 0.6. As the cells synthesize luteolin in the bioreactor, RFP expression levels increase as cell stress increases. RFP expression levels will be measured by the fluorescence sensor. When RFP expression levels reach 900 (as measured using a microplate reader (H1, Biotek, USA)), the blue light is turned on. Once the RFP expression levels are acceptable, the blue light is turned off. This cycle repeats.


Testing

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 in the real system, and hence would not interfere with each other. We were thus able to calibrate OD and fluorescence separately.


  1. Prepare 20 ml of E.coli cells harbouring Brep-RFP-YbaQ at OD = 3. These cells should have been previously been continuously exposed to blue light to repress RFP expression.
  2. Dilute the cell medium to get 12 different samples with ODs ranging from 0.1 to 3. Label them from 1 - 12.
  3. Measure the actual OD of sample 1 using the NanoDrop.
  4. Transfer sample 1 into the cuvette with a syringe. Place the cuvette into the casing and close it.
  5. Using PLX-DAQ, obtain approximately 10 readings of TSL235R’s output frequency when the 600 nm LED is lit.
  6. Repeat steps 3-5 for the rest of the samples.
  7. Plot a graph of output frequency against OD readings from the NanoDrop.


  1. Prepare 20 ml of E.coli cells harbouring Brep-RFP-YbaQ at RFP reading = 7100.
  2. Dilute the cell medium to get 12 different samples with RFP readings ranging from 100 to 7100. Label them from 1-12.
  3. Measure the actual RFP reading of sample 1 using the NanoDrop.
  4. Transfer sample 1 into the cuvette with a syringe. Place the cuvette into the casing and close it.
  5. Using PLX-DAQ, obtain approximately 10 readings of TSL235R’s output frequency when the 535 nm LED is lit.This LED excites the RFP, causing it to fluoresce red with a wavelength of 600nm.
  6. Repeat steps 3-5 for the rest of the samples.
  7. Plot a graph of output frequency against RFP readings from the NanoDrop.

By following our experimental procedure, we were able to plot calibration curves for OD and fluorescence sensing. Please visit Results:Cell-Machine Interface for the results of our characterization of this sensor.

Construction

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!


  • Arduino Uno x 1
  • 600 nm LED x 1
  • 535 nm LED x 1
  • Light-to-frequency converter TSL235R x 1
  • LEE filter (peak transmission 600 nm)
  • OD=6 mm ID=3 mm acrylic tubes x 1 m
  • Acrifix 1R 0912 UV Adhesive


Video
Figure 4. Cuvette assembly


Video
Figure 5. Sensor Assembly


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.



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We’re a little disruptive, dangerous and maybe a tad too bold. But the world needs some shaking up every now and then, and we think we’re just the right people for that.

Together we’re going to engineer biology and create something awesome.

CONTACT US

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