Difference between revisions of "Team:Lethbridge/InterLab"

 
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<h1>INTERLAB STUDY</h1>
 
<h1>INTERLAB STUDY</h1>
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<p class="f14">Synthetic biology interconnects the principles of biology and engineering. Reliable and repeatable measurement is one of the fundamentals of engineering, which synthetic biology must also aspire to achieve. However, replicability of fluorescence measurements between labs presents a unique challenge because of differences in how the data are acquired and processed, and other sources of systematic variability that are not accounted for (such as the number of cells in a sample). By using “relative expression” comparisons, users from different labs cannot directly compare results. <br><br>
+
<p class="f12">Synthetic biology interconnects the principles of biology and engineering. Reliable and repeatable measurement is one of the fundamentals of engineering, which synthetic biology must also aspire to achieve. However, replicability of fluorescence measurements between labs presents a unique challenge because of differences in how the data are acquired and processed, and other sources of systematic variability that are not accounted for (such as the number of cells in a sample). By using “relative expression” comparisons, users from different labs cannot directly compare results. <br><br>
 
The purpose of the 2018 InterLab Study was to attempt to reduce lab-to-lab variability by standardizing green fluorescent protein (GFP) fluorescence measurements. This was done by normalizing GFP fluorescence to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).</p>
 
The purpose of the 2018 InterLab Study was to attempt to reduce lab-to-lab variability by standardizing green fluorescent protein (GFP) fluorescence measurements. This was done by normalizing GFP fluorescence to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).</p>
 
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<h3>Overview</h3>
 
<h3>Overview</h3>
<p class="f14"> Two approaches were used:<br><br>
+
<p class="f12"> Two approaches were used:<br><br>
  
<b>1. Converting between absorbance of cells to absorbance of a known concentration of beads</b><br>
+
<b>1. Converting between absorbance of cells to absorbance of a known concentration of beads.</b><br>
Absorbance measurement uses the light scattering by a sample of cells in liquid.The variable in this measurement is the concentration of the cell. With assumption that the silica beads at the size of typical E.coli cells would scatter the light in similar way, the absorbance measurement with known concentration of the beads can be standardized as the “equivalent concentration of beads” measurement.  
+
Absorbance is a measure of how light is scattered by cells in liquid, which can be used to estimate the concentration of cells in the sample. Assuming that silica beads approximately the same size and shape of <i>E. coli</i> cells would have similar optical properties, we can use the absorbance measurement of a known concentration of beads to produce a standardized “equivalent concentration of beads” measurement.  
 
<br><br>
 
<br><br>
 
<b>2. Counting colony-forming units (CFUs) from the sample.</b><br>
 
<b>2. Counting colony-forming units (CFUs) from the sample.</b><br>
    Simply, growing cells on a plate and counting the number of colonies on the plate can determine the amount of cells in a sample of liquid. The concentration of the sample can be obtained with the number of singly grown cells in the plate, which indicates the amount of cells, and the volume of media that is plated out. The positive and negative control samples will be used to calculate a conversion factor from absorbance to CFU.<br><br>
+
We can estimate the number of individual cells in a volume of liquid media by plating a sample and quantifying colonies (which are each clones of a single cell). The number of colonies in positive and negative control samples can be used to calculate a conversion factor from absorbance to CFU.<br><br>
 
For complete methods, refer to: <a href="https://2018.igem.org/Measurement/InterLab/Plate_Reader"> https://2018.igem.org/Measurement/InterLab/Plate_Reader</a></p><br><br>
 
For complete methods, refer to: <a href="https://2018.igem.org/Measurement/InterLab/Plate_Reader"> https://2018.igem.org/Measurement/InterLab/Plate_Reader</a></p><br><br>
  
 
<h3>Our Equipment</h3>
 
<h3>Our Equipment</h3>
  
<p class="f14">A Molecular Devices Spectramax i3x plate reader was used to measure sample absorbance and fluorescence. This device has variable temperature settings, top optics, and pathlength correction, which can be disabled. All GFP measurements were taken at wavelengths of 535/25nm for emission and 485/20nm for excitation. We used clear-bottomed 96-well plates for all measurements.</p><br><br>
+
<p class="f12">A Molecular Devices Spectramax i3x plate reader was used to measure sample absorbance and fluorescence. This device has variable temperature settings, top optics, and pathlength correction, which can be disabled. All GFP measurements were taken at wavelengths of 535/25nm for emission and 485/20nm for excitation. We used clear-bottomed 96-well plates for all measurements.</p><br><br>
  
  
 
<h3> Calibration </h3>
 
<h3> Calibration </h3>
  
<p class="f14">1. LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a
+
<p class="f12">1. LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a
 
conversion factor to transform absorbance data into a comparable OD measurement.<br><br>
 
conversion factor to transform absorbance data into a comparable OD measurement.<br><br>
  
2. A dilution series of monodisperse silica microspheres was used to measure absorbance and produce a standard curve of particle concentration. The size and optical characteristics of these microspheres are similar to cells and there is a known amount of particles per volume. This standard curve was later used to convert absorbance measurements to an estimated number of cells.<br><br>
+
2. A dilution series of monodisperse silica microspheres was used to measure absorbance and produce a standard curve of particle concentration. This standard curve was later used to convert absorbance measurements to an estimated number of cells.<br><br>
  
3. A dilution series of fluorescein, which has similar excitation and emission properties to GFP, was used to generate a fluorescein standard curve. This standard curve was later used to convert
+
3. A dilution series of fluorescein, which has similar excitation and emission properties to GFP, was used to generate a fluorescein standard curve. This standard curve was later used to convert cell-based readings to an equivalent fluorescein concentration.
Cell-based readings to an equivalent fluorescein concentration.
+
 
</p><br></br>
 
</p><br></br>
  
 
<h3> Cell Measurement Protocol </h3>
 
<h3> Cell Measurement Protocol </h3>
  
<p class="f14">1. Measured a standard curve for fluorescein to allow this data to be standardized with the data from other iGEM labs. Fluorescein displays similar excitation and emission as GFP and could also be read by the plate reader. This allowed for measurements of GFP fluorescing cells to to be transformed into similar fluorescein readings. </p><br></br>
+
<p class="f12">Briefly, we measured the raw absorbance and fluorescence of diluted cells (transformed with the below testing devices) at 0 hours and after 6 hours of incubation at 37°C.</p><br></br>
<p class="f14">2. Followed a LUDOX protocol to serve as a reference point that functioned as a conversion factor. This conversion factor allowed for the absorbance values taken at 600 nm to be converted into OD600 measurements  </p><br></br>
+
<p class="f14">3. A plate was prepared with silica microspheres and read by the plate reader at 600 nm. These microspheres have similar functional characteristics to cells that allows for the absorbance measurements to accurately estimate cell counts. </p><br></br>
+
  
<h3> Testing Devices </h3>
 
  
<p class="f14"> The negative control did not contain fluorescent DNA parts and positive control is the fluorescent beads with known value. From the fluorescence values of two controls, the fluorescence value of each devices were calculated. Additionally, the kit plate and well locations were also pre-determined to minimize the detecting errors between trials. </p>
 
 
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<table style="width:70%">
   <tr>
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   <tr class="f11">
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Device</th>
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     <th font-weight: bold;">Device</th>
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Pert number</th>
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     <th font-weight: bold;">Part number</th>
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Plate</th>  
+
     <th font-weight: bold;">Plate</th>  
     <th style="background-color: #80471C; color: #ffffff; font-weight: bold;">Location</th>
+
     <th font-weight: bold;">Location</th>
 
   </tr>
 
   </tr>
<tr>
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<tr class="f10">
 
     <td>Negative Control</td>
 
     <td>Negative Control</td>
 
     <td>BBa_R0040</td>
 
     <td>BBa_R0040</td>
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     <td>Well 2D</td>
 
     <td>Well 2D</td>
 
   </tr>
 
   </tr>
<tr>
+
<tr class="f10">
 
     <td>Positive Control</td>
 
     <td>Positive Control</td>
 
     <td>BBa_I20270</td>
 
     <td>BBa_I20270</td>
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     <td>Well 2B</td>
 
     <td>Well 2B</td>
 
   </tr>
 
   </tr>
  <tr>
+
<tr class="f10">
 
     <td>Test Device 1 </td>
 
     <td>Test Device 1 </td>
 
     <td>BBa_J364000</td>
 
     <td>BBa_J364000</td>
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     <td>Well 2F</td>
 
     <td>Well 2F</td>
 
   </tr>
 
   </tr>
  <tr>
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<tr class="f10">
 
     <td>Test Device 2</td>
 
     <td>Test Device 2</td>
 
     <td>BBa_J364001</td>
 
     <td>BBa_J364001</td>
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     <td>Well 2H</td>
 
     <td>Well 2H</td>
 
   </tr>
 
   </tr>
<tr>
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<tr class="f10">
 
     <td>Test Device 3</td>
 
     <td>Test Device 3</td>
 
     <td>BBa_J364002</td>
 
     <td>BBa_J364002</td>
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     <td>Well 2J</td>
 
     <td>Well 2J</td>
 
   </tr>
 
   </tr>
<tr>
+
<tr class="f10">
 
     <td>Test Device 4</td>
 
     <td>Test Device 4</td>
 
     <td>BBa_J364007</td>
 
     <td>BBa_J364007</td>
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     <td>Well 2L</td>
 
     <td>Well 2L</td>
 
   </tr>
 
   </tr>
<tr>
+
<tr class="f10">
 
     <td>Test Device 5</td>
 
     <td>Test Device 5</td>
 
     <td>BBa_J364008</td>
 
     <td>BBa_J364008</td>
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     <td>Well 2N</td>
 
     <td>Well 2N</td>
 
   </tr>
 
   </tr>
<tr>
+
<tr class="f10">
 
     <td>Test Device 6</td>
 
     <td>Test Device 6</td>
 
     <td>BBa_J364009</td>
 
     <td>BBa_J364009</td>
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<h1>Results</h1>
 
<h1>Results</h1>
  
<p class="f14">We expected a linear relationship between concentration and fluorescence/absorbance. However, the log-scale particle standard curve (Figure 2B) does not exhibit a linear relationship. Possible sources of error include pipetting error or oversaturation of the detector.<br><br>
+
<div class="oneText-Wrapper">
The conversion of average fluorescence to reference unit was designed to reduce the impact of unorganized unit system.
+
<div class="OneText-Text">
<br>For fluorescence to OD conversion, fluorescence was converted to absorbance and to OD.
+
<p class="f12">In our standard curves, we expected a linear relationship between concentration and fluorescence/absorbance. However, the log-scale particle standard curve (Figure 2B) does not exhibit a linear relationship. Possible sources of error include pipetting error or oversaturation of the detector.</p><br><br>
<br>For fluorescence to particle conversion, Molecules of Equivalent of Fluorescence (MEFL) was converted to absorbance and to particles.
+
<br>The trend showed that except device 3, all other devices contained fluorescence part and the device 2 and 5 had increased fluorescence part after 6 hours, but device 1,4, and 6 had decreased fluorescence part after 6 hours. </p>
+
 
     </div>
 
     </div>
 
</div>
 
</div>
 +
 
<div style="clear:both"></div>
 
<div style="clear:both"></div>
  
<h3>Fluorescein Standard Curve</h3>
+
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<div class="twoText-Wrapper">
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    <div class="twoText-Text">
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<img src="https://static.igem.org/mediawiki/2018/c/c4/T--Lethbridge--Fluorescein_Standard_Curve_V2.png" alt="">
        <center><img src="https://static.igem.org/mediawiki/2018/c/c4/T--Lethbridge--Fluorescein_Standard_Curve_V2.png" width = 400 px; height=250px; padding: 30px;></center>
+
</div>
        <p>Fluorescein standard curve</p>
+
<div class="twoImage-Image">
    </div>
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<img src="https://static.igem.org/mediawiki/2018/9/9c/T--Lethbridge--Fluorescein_Standard_Curve_log_scale_V2.png" alt="">
    <div class="twoText-Text">
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</div>
      <center><img src="https://static.igem.org/mediawiki/2018/9/9c/T--Lethbridge--Fluorescein_Standard_Curve_log_scale_V2.png" width = 400 px; height=250px; padding: 30px;></center>
+
    <p>Fluorescein standard curve (log scale)</p>
+
    </div>
+
 
</div>
 
</div>
<div style="clear:both"></div>
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<div style="clear: both"></div>
<br><br>
+
  
<h3>Particle Standard Curve</h3>
+
 
<div class="twoText-Wrapper">
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<div class="oneText-Wrapper">
    <div class="twoText-Text">
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<div class="oneText-Text">
        <center><img src="https://static.igem.org/mediawiki/2018/6/6a/T--Lethbridge--Particle_Standard_Curve_V2.png" width = 400 px; height=250px; padding: 30px;></center>
+
<p class="f10"><b>Figure 1:</b> A) Fluorescein standard curve. B) Fluorescein standard curve (log-scale).</p>
        <p>Standard curve of particles</p>
+
</div>
    </div>
+
    <div class="twoText-Text">
+
      <center><img src="https://static.igem.org/mediawiki/2018/9/9d/T--Lethbridge--Particle_Standard_Curve_log_scale_V2.png"width = 400 px; height=250px; padding: 30px;></center>
+
    <p>Standard curve of particles (log scale)</p>
+
    </div>
+
 
</div>
 
</div>
<div style="clear:both"></div>
+
<div style="clear: both"></div><br><br>
<br><br>
+
  
<h3>Average Fluorescence</h3>
+
 
<div class="twoText-Wrapper">
+
 
    <div class="twoText-Text">
+
 
        <center><img src="https://static.igem.org/mediawiki/2018/9/99/T--Lethbridge--Average_Fluorescence_vs_OD.png" width = 400 px; height=250px; padding: 30px;></center>
+
<div class="twoImage-Wrapper">
        <p>Average fluorescence per OD</p>
+
<div class="twoImage-Image">
    </div>
+
<img src="https://static.igem.org/mediawiki/2018/6/6a/T--Lethbridge--Particle_Standard_Curve_V2.png" alt="">
    <div class="twoText-Text">
+
</div>
      <center><img src="https://static.igem.org/mediawiki/2018/2/2f/T--Lethbridge--Average_Fluorescence_vs_particle.png" width = 400 px; height=250px; padding: 30px;></center>
+
<div class="twoImage-Image">
    <p>Average fluorescence per particle  </p>
+
<img src="https://static.igem.org/mediawiki/2018/9/9d/T--Lethbridge--Particle_Standard_Curve_log_scale_V2.png" alt="">
    </div>
+
</div>
 
</div>
 
</div>
<div style="clear:both"></div>
+
<div style="clear: both"></div>
<br><br>
+
 
 +
 
 +
<div class="oneText-Wrapper">
 +
<div class="oneText-Text">
 +
<p class="f10"><b>Figure 2:</b> A) Particle standard curve. B) Particle standard curve (log-scale).</p>
 +
</div>
 +
</div>
 +
<div style="clear: both"></div><br><br>
 +
 
 +
<div class="twoImage-Wrapper">
 +
<div class="twoImage-Image">
 +
<img src="https://static.igem.org/mediawiki/2018/9/99/T--Lethbridge--Average_Fluorescence_vs_OD.png" alt="">
 +
</div>
 +
<div class="twoImage-Image">
 +
<img src="https://static.igem.org/mediawiki/2018/2/2f/T--Lethbridge--Average_Fluorescence_vs_particle.png" alt="">
 +
</div>
 +
</div>
 +
<div style="clear: both"></div>
 +
 
 +
 
 +
<div class="oneText-Wrapper">
 +
<div class="oneText-Text">
 +
<p class="f10"><b>Figure 3:</b> A) Average fluorescence per optical density. B) Average fluorescence per particle.</p>
 +
</div>
 +
</div>
 +
<div style="clear: both"></div><br><br>
  
  

Latest revision as of 23:33, 17 October 2018



Interlab Banner Image

INTERLAB STUDY

Synthetic biology interconnects the principles of biology and engineering. Reliable and repeatable measurement is one of the fundamentals of engineering, which synthetic biology must also aspire to achieve. However, replicability of fluorescence measurements between labs presents a unique challenge because of differences in how the data are acquired and processed, and other sources of systematic variability that are not accounted for (such as the number of cells in a sample). By using “relative expression” comparisons, users from different labs cannot directly compare results.

The purpose of the 2018 InterLab Study was to attempt to reduce lab-to-lab variability by standardizing green fluorescent protein (GFP) fluorescence measurements. This was done by normalizing GFP fluorescence to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).



Methods

Overview

Two approaches were used:

1. Converting between absorbance of cells to absorbance of a known concentration of beads.
Absorbance is a measure of how light is scattered by cells in liquid, which can be used to estimate the concentration of cells in the sample. Assuming that silica beads approximately the same size and shape of E. coli cells would have similar optical properties, we can use the absorbance measurement of a known concentration of beads to produce a standardized “equivalent concentration of beads” measurement.

2. Counting colony-forming units (CFUs) from the sample.
We can estimate the number of individual cells in a volume of liquid media by plating a sample and quantifying colonies (which are each clones of a single cell). The number of colonies in positive and negative control samples can be used to calculate a conversion factor from absorbance to CFU.

For complete methods, refer to: https://2018.igem.org/Measurement/InterLab/Plate_Reader



Our Equipment

A Molecular Devices Spectramax i3x plate reader was used to measure sample absorbance and fluorescence. This device has variable temperature settings, top optics, and pathlength correction, which can be disabled. All GFP measurements were taken at wavelengths of 535/25nm for emission and 485/20nm for excitation. We used clear-bottomed 96-well plates for all measurements.



Calibration

1. LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a conversion factor to transform absorbance data into a comparable OD measurement.

2. A dilution series of monodisperse silica microspheres was used to measure absorbance and produce a standard curve of particle concentration. This standard curve was later used to convert absorbance measurements to an estimated number of cells.

3. A dilution series of fluorescein, which has similar excitation and emission properties to GFP, was used to generate a fluorescein standard curve. This standard curve was later used to convert cell-based readings to an equivalent fluorescein concentration.



Cell Measurement Protocol

Briefly, we measured the raw absorbance and fluorescence of diluted cells (transformed with the below testing devices) at 0 hours and after 6 hours of incubation at 37°C.



Device Part number Plate Location
Negative Control BBa_R0040 Kit Plate 7 Well 2D
Positive Control BBa_I20270 Kit Plate 7 Well 2B
Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
Test Device 6 BBa_J364009 Kit Plate 7 Well 2P


Results

In our standard curves, we expected a linear relationship between concentration and fluorescence/absorbance. However, the log-scale particle standard curve (Figure 2B) does not exhibit a linear relationship. Possible sources of error include pipetting error or oversaturation of the detector.



Figure 1: A) Fluorescein standard curve. B) Fluorescein standard curve (log-scale).



Figure 2: A) Particle standard curve. B) Particle standard curve (log-scale).



Figure 3: A) Average fluorescence per optical density. B) Average fluorescence per particle.