Difference between revisions of "Team:TokyoTech/InterLab"

 
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      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">InterLab</h2>
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<h2 id="Introduction" class="smalltitle">1. Introduction</h2>
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　　　　　　<p class="text">In iGEM 2015, the Interlab Study was held, where we measured the expression level of GFP using three designated devices. It was the first time for our team to join this Interlab Study. In addition to the three designated devices, we also measured the expression level of GFP from a positive control and a negative control using the flow cytometer and the plate reader. Also, for the plate reader, we succeeded in calculating the absolute unit by drawing the calibration curve using sodium fluorescein.</p>
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          <h2 id="Summary" class="smalltitle">2. Summary of the Experiment</h2>
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　　　　　　<p class="text">Our purpose was to obtain the fluorescence data of the three designated devices and to compare them. We prepared Device1〜Device3, Positive control and Negative control as shown below. We measured the exact same colonies of the exact same samples with both the plate reader and the flow cytometer.</p>
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          <li><p class="list">Device 1: J23101 + I13504(pSB1C3)</p></li>
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          <li><p class="list">Device 2: J23106 + I13504(pSB1C3)</p></li>
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          <li><p class="list">Device 3: J23117 + I13504(pSB1C3)</p></li>
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          <li><p class="list">Positive control: BBa_I20270(pSB1C3)</p></li>
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      <h4 align="center" class="fig"><strong>Fig.3-6-2-1.</strong>&nbsp;designated devices</h4>
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          <h2 id="Results" class="smalltitle">3. Results</h2>
 
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              <h3 id="Plate" class="sub5">3.1. Plate reader</h3>
 
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　　　　　　    <p class="text2">First of all, we calibrated our plate reader by confirming the linear relationship between sodium fluorescein concentration and fluorescence (Fig. 3-6-3-1). The way we obtained the calibration curve is descried in the 4. Material and Method section.
 
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                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/c/c7/Tokyo_Tech_Interlab_Fig.3-7-3-1.png" />
 
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                <h4 align="center" class="fig"><strong>Fig.3-6-3-1.</strong>&nbsp;Calibration curve</h4>
 
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　　　　　　    <p class="text2">We measured three colonies(#1〜3) three times (Technical replicate 1〜3) per each sample (Device1〜3, positive control and negative control).<br>
 
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&nbsp;&nbsp;Using the calibration curve (Fig. 3-6-3-1), we were able to obtain the absolute unit of fluorescence (Table. 3-6-3-1). The way we obtained the absolute unit is described in the 4. Material and Method section. These results show that the intensity of fluorescence was in the following order, Device1>Device2>positive control>Device3>negative control.</p><br>
 
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                <h4 align="center" class="fig"><strong>Table. 3-6-3-1.</strong>&nbsp;The absolute unit of fluorescence intensity</h4>
 
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                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/c/ca/Tokyo_Tech_Interlab_Table.3-7-3-1.png" width="940px"/>
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    <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Introduction</b></h2>
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<p class="text2">We calculated the arithmetic mean for each sample by adding the nine values of all three colonies and dividing it by 9.
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We also calculated the standard deviation for each sample from the calculated arithmetic mean. (Table. 3-6-3-2)
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                <h4 align="center" class="fig"><strong>Table. 3-6-3-2.</strong>&nbsp;Arithmetic mean (Mean) and Standard deviation (S.D) of samples.</h4>
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    <p style="font-size: 16px; text-indent:1em">In iGEM 2017, the InterLab Study is one of the bronze medal criteria. We measured the GFP expression level of the six designated devices, positive control and negative control using the plate reader. It was the second time for our team to join this InterLab Study (The first time in iGEM 2015).  
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                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a6/Tokyo_Tech_Interlab_Table.3-7-3-2.png" width="700px"/>
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      <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/c/c3/Tokyo_Tech_Interlab_Fig.3-7-3-2.png" width="530px"/>
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      <h4 align="center" class="fig"><strong>Fig.3-6-3-2.</strong>&nbsp;Results from the plate reader
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<br>The error bar represents the standard deviation for each sample calculated from the nine values of all three colonies.</h4>
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              <h3 id="Flow" class="sub5">3.2. Flow cytometer</h3>
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　　　　　　    <p class="text2">We measured the geometric mean of fluorescence intensity for each sample. The results are shown below (Table.3-6-3-3). We measured three colonies(#1〜3) three times (Technical replicate 1〜3) per each sample (Device1〜3,positive control and negative control).<br>
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&nbsp;&nbsp;These results show that the intensity of fluorescence was in the following order, Device1>Device2>positive control>Device3>negative control. (Table. 3-6-3-4)(Fig. 3-6-3-3)<br>
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&nbsp;&nbsp;These results are the same as the results from the measurement done by the plate reader.</p><br>
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                <h4 align="center" class="fig"><strong>Table. 3-6-3-3.</strong>&nbsp;Results from the flow cytometer</h4>
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                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/80/Tokyo_Tech_Interlab_Table.3-7-3-3.png" width="940px"/>
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                <h4 align="center" class="fig"><strong>Table. 3-6-3-4.</strong>&nbsp;Arithmetic mean (Mean) and Standard deviation (S.D) of samples.</h4>
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                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/0a/Tokyo_Tech_Interlab_Table.3-7-3-4.png" width="700px"/>
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Summary</b></h2>
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      <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/3e/Tokyo_Tech_Interlab_Fig.3-7-3-4.png" width="530px"/>
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      <h4 align="center" class="fig"><strong>Fig.3-6-3-3.</strong>&nbsp;Results from the flow cytometer　<br>The error bar represents the standard deviation for each sample calculated from the nine values of all three colonies.
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      </table><br><br>
+
  
−
          <h2 id="Materials" class="smalltitle">4. Materials and Methods</h2>
+
    <p style="font-size: 16px; text-indent:1em">
−
              <h3 id="Const" class="sub5">4.1.  Construction</h3>
+
  Our purpose was to investigate the RBS intensity and promoter intensity effects on gene expression by obtaining the fluorescence data of samples and comparing them. We prepared Device1〜Device6, Positive Control and Negative Control as shown below.
−
              <h3 class="sub5">-Strain</h3>
+
    </p><br>
−
　　　　　　    <p class="text2">All the samples were DH5alpha strain.</p>
+
 
−
              <h3 class="sub5">-Plasmids</h3>
+
    <!-- 図開始 -->
−
　　　　　　    <p class="text2">Device 1: J23101 + I13504(pSB1C3)
+
    <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
−
                <table width="980 px" border="0px">
+
      <figure>
−
                  <tr>
+
      <img src="https://static.igem.org/mediawiki/parts/2/2f/T--TokyoTech--Interlab_Devices.png" style="max-width:70%">
−
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/1/1c/Tokyo_Tech_Device1.png"/>
+
       <figcaption style="font-size: 16px">Fig. 1 Designated devices</figcaption>
−
      </td>
+
       </figure>
−
      </tr>
+
       </div><!-- 図終了 -->
−
      <tr>
+
 
−
      <td width="980px">
+
  </div>
−
       <h4 align="center" class="fig"><strong>Fig.3-6-4-1.</strong></h4>
+
 
−
       <td>
+
<hr>
−
       </tr>
+
 
−
      </table><br>
+
<div class="w3-container" id="overview" style="margin-top:20px">
−
　　　　　　    <p class="text2">Device 2: J23106 + I13504(pSB1C3)
+
    <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Results</b></h2>
−
                <table width="980 px" border="0px">
+
    <hr style="width:50px;border:5px solid red" class="w3-round">
−
                  <tr>
+
 
−
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/3b/Tokyo_Tech_Device2.png"/>
+
    <p style="font-size: 16px; text-indent:1em">
−
      </td>
+
    1. We obtained LUDOX correction values.</p></br>
−
      </tr>
+
    <!-- 図開始 -->
−
      <tr>
+
    <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
−
      <td width="980px">
+
      <figure>
−
      <h4 align="center" class="fig"><strong>Fig.3-6-4-2.</strong></h4>
+
      <img src="https://static.igem.org/mediawiki/2017/4/4e/T--TokyoTech--LUDOX_interlab.png" style="max-width:30%">
−
      <td>
+
       <figcaption style="font-size: 16px">Fig. 2 LUDOX correct value </figcaption>
−
      </tr>
+
       </figure>
−
      </table><br>
+
       </div><!-- 図終了 -->
−
　　　　　　    <p class="text2">Device 3: J23117 + I13504(pSB1C3)
+
<p style="font-size: 16px; text-indent:1em">
−
                <table width="980 px" border="0px">
+
2. We obtained the two Fluorescein Standard Curve (normal and log scale).</p></br>
−
                  <tr>
+
<!-- 図開始 -->
−
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/b/be/Tokyo_Tech_Device3.png"/>
+
    <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
−
      </td>
+
      <figure>
−
      </tr>
+
      <img src="https://static.igem.org/mediawiki/2017/8/82/T--TokyoTech--Fluorescein_Standard_Curve.png" style="max-width:60%">
−
      <tr>
+
       <figcaption style="font-size: 16px">Fig. 3 Fluorescein Standard Curve</figcaption>
−
      <td width="980px">
+
       </figure>
−
       <h4 align="center" class="fig"><strong>Fig.3-6-4-3.</strong></h4>
+
       </div><!-- 図終了 -->
−
      <td>
+
     
−
       </tr>
+
      <!-- 図開始 -->
−
       </table><br>
+
    <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
−
　　　　　　    <p class="text2">Positive control: BBa_I20270(pSB1C3)
+
      <figure>
−
                <table width="980 px" border="0px">
+
      <img src="https://static.igem.org/mediawiki/2017/a/ad/T--TokyoTech--Fluorescein_Standard_Curve%28log_scale%29.png" style="max-width:60%">
−
                  <tr>
+
       <figcaption style="font-size: 16px">Fig. 4 Fluorescein Standard Curve (log scale)</figcaption>
−
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/09/Tokyo_Tech_Interpositive.png"/>
+
       </figure>
−
      </td>
+
       </div><!-- 図終了 -->
−
      </tr>
+
<p style="font-size: 16px; text-indent:1em">
−
      <tr>
+
3. We obtained the graph bellow. The RFU of GFP / Turbidity was in the following order, Device1>Device4>Device2>Positive Control>Device5>Device3>Device6>Negative Control.
−
      <td width="980px">
+
    </p><br>
−
       <h4 align="center" class="fig"><strong>Fig.3-6-4-4.</strong></h4>
+
 
−
      <td>
+
       
−
       </tr>
+
     
−
       </table><br>
+
      <!-- 図開始 -->
−
　　　　　　    <p class="text2">Negative control: BBa_R0040(pSB1C3)
+
    <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
−
                <table width="980 px" border="0px">
+
      <figure>
−
                  <tr>
+
      <img src="https://static.igem.org/mediawiki/2017/f/f3/T--TokyoTech--Fluo--OD600ave.png" style="max-width:70%">
−
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/1/1b/Tokyo_Tech_Internegative.png"/>
+
      <figcaption style="font-size: 16px">Fig. 5 Fluorescein (using AVERGE)</figcaption>
−
      </td>
+
      </figure>
−
      </tr>
+
      </div><!-- 図終了 -->
−
      <tr>
+
     
−
      <td width="980px">
+
      <!-- 図開始 -->
−
       <h4 align="center" class="fig"><strong>Fig.3-6-4-5.</strong></h4>
+
    <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
−
      <td>
+
      <figure>
−
       </tr>
+
      <img src="https://static.igem.org/mediawiki/2017/a/a5/T--TokyoTech--Fluo--OD600geo.png" style="max-width:70%">
−
       </table><br>
+
       <figcaption style="font-size: 16px">Fig. 6 Fluorescein (using GEOMEAN)</figcaption>
−
              <h3 class="sub5">-Sequence Data</h3>
+
      </figure>
−
　　　　　　    <p class="text2">Please refer to <a href ="https://2015.igem.org/Team:Tokyo_Tech/Experiment/Interlab/Sequence">Sequence Data</a> page.<br>
+
      </div><!-- 図終了 -->
−
              <h3 id="ID" class="sub5">4.2. Instruments and Date</h3>
+
     
−
              <h3 id="Inst" class="sub6">4.2.1. Instruments</h3>
+
 
−
                <p class="text2"><strong>-Plate reader</strong></p>
+
  </div>
−
                  <p class="text3">We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJIFilm Life Science. The wavelength of light we used to excite the cells was 473 nm. We used BPB1 (530DF20) filter to capture the light emission from the cells. The sampling frequency is only one time.
+
 
−
</p>
+
  <hr>
−
                <p class="text2"><strong>-Flow cytometer</strong></p>
+
 
−
                  <p class="text3">We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company. The wavelength of light we used to excite the cells was 488 nm. We used laser detection channel FL1 to capture the light emission from the cells. Laser detection channel Fl1 was used with sensitivity 680 [v]. The sampling frequency is only one time.</p>
+
<div class="w3-container" id="overview" style="margin-top:20px">
−
              <h3 id="Date" class="sub6">4.2.2. Date</h3>
+
    <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h2>
−
                <p class="text3">Cloning of constructs was confirmed by August 21st 2015. Transformant plates were from 24 August 2015. All the samples were measured on August 27th 2015.</p>
+
    <hr style="width:50px;border:5px solid red" class="w3-round">
−
              <h3 id="Protocol" class="sub5">4.3. Protocol</h3>
+
 
−
              <h3 id="reader" class="sub6">4.3.1. Plate reader</h3>
+
    <p style="font-size: 16px; text-indent:1em">
−
                    <p class="text4">
+
  Judging from the results, the gene expression is controlled by the intensity of promoter and the intensity of RBS. The promoter intensity of is supposed to be in the following order, J23101>J23106>J23117. The RBS intensity of I13504 is supposed to be higher than that of BCD2.E0040. Moreover, it is considered that the GFP expression level is dependent on individual to some extent.
−
1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3 mL LB medium containing chloramphenicol (35 microg / mL) at 37°C for 17 h and shake at 180 rpm.<br>
+
   </p><br>
−
2. Measured the OD590 of each sample and diluted each sample to adjust OD590 within 5% of 0.5.<br>
+
−
3. Set the plate reader to measure GFP.<br>  
+
−
4. Take 1 mL of the samples, and centrifuge at 9000x g, 1 min, 4°C.<br>
+
−
5. Remove the supernatants by using P1000 pipette. <br>
+
−
6. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspended.<br>
+
−
7. Place 200 microL of each sample into the 96-well plate as described in Table. 3-6-4-1.<br>
+
−
8. Measure the fluorescence intensity with plate reader.<br>
+
−
9. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.<br></p>
+
−
                <table width="940 px" border="0px">
+
−
                <tr>
+
−
                <td width="940px">
+
−
                <h4 align="center" class="fig"><strong>Table. 3-6-4-1.</strong>&nbsp;Position of samples in 96-well plate</h4>
+
−
                <td>
+
−
                </tr>
+
−
                <tr>
+
−
                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f7/Tokyo_Tech_Interlab_Table.3-7-4-1.png" width="700px"/>
+
−
                </td>
+
−
                </tr>
+
−
       </table><br>
+
−
              <h3 id="meter" class="sub6">4.3.2. Flow cytometer</h3>
+
−
                    <p class="text4">
+
−
1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3 mL LB medium containing chloramphenicol (35 microg / mL) at 37°C for 17 h and shake at 180 rpm.<br>
+
−
2. Start preparing the flow cytometer 1 h before the end of incubation.<br>
+
−
3. Measure the OD590 and adjust the volume of each sample to centrifuge so that the amount of pellet will be about the same for every sample.<br>
+
−
4. Centrifuge the samples at 9000x g, 1 min, 4°C.<br>
+
−
5. Remove the supernatants by using P1000 pipette and suspend the samples with 1 mL of filtered PBS (phosphate-buffered saline).<br>
+
−
6. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
+
−
7. Measure fluorescence intensity with flow cytometer.<br><br>
+
−
              <h3 id="How" class="sub5">4.4. How to process the data</h3>
+
−
              <h3 id="Pr" class="sub6">4.4.1. Plate reader</h3>
+
−
                    <p class="text4">
+
−
<strong>-How to draw the calibration curve</strong><br>
+
−
1. Place 200 microL of various concentrations of sodium fluorescein (500, 375, 250, 125, 50, 25, 10, 5 ng / mL and PBS only) into the 96-well plate in triplicate.<br>
+
−
2. Measure the fluorescence intensity with the plate reader.<br>
+
−
3. Rotate the 96-well plate 180 degrees horizontally.<br>
+
−
4. Measure the fluorescence intensity again.<br>
+
−
5. Determine the auto-fluorescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence. <br>
+
−
6. Subtract background fluorescence from each fluorescence intensity value of each well containing sodium fluorescein.<br>
+
−
7. Take the arithmetic mean of the three technical replicates of sodium fluorescein of each concentration.<br>
+
−
8. Draw the calibration curve.<br> </p><br>
+
−
                    <p class="text4">
+
−
<strong>-How to obtain the absolute unit of fluorescence intensity</strong><br>
+
−
1. Measure the fluorescence intensity with the plate reader.<br>
+
−
2. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.<br>
+
−
3. Calculate the arithmetic mean of these two results.<br>
+
−
4. Determine the auto-florescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence.<br>  
+
−
5. Subtract the background fluorescence from each well containing the samples.<br>
+
−
6. Divide them by the value of OD590 of each sample.<br>
+
−
7. Calculate the ng / mL fluorescence per OD590 unit by the formula we obtained from drawing the calibration curve.<br>
+
−
              <h3 id="Flow cytometer" class="sub6">4.4.2. Flow cytometer</h3>
+
−
                    <p class="text3">Cells were gated according to the side scatter (SSC) and the forward scatter (FCS) to exclude cell debris and impurities.</p><br><br>
+
−
              <h3 id="Individuals" class="sub5">4.5. Individuals responsible for conducting Interlab study</h3>
+
−
　　　　　　   <p class="text2">
+
−
Misa Minegishi : Measured the devices and processed the data.<br>
+
−
&nbsp;&nbsp;&nbsp;Yuta Yamazaki : Measured the devices and processed the data.<br>
+
−
&nbsp;&nbsp;&nbsp;Hiraku Tokuma : Created the devices.<br>
+
−
&nbsp;&nbsp;&nbsp;Riku Shinohara: Created the devices.<br>
+
−
          <h2 id="Reference" class="smalltitle">5. Reference</h2>
+
−
　　　　　　<p class="text"><a href ="https://2014.igem.org/Team:Imperial/InterLab_Study">2014 Imperial Interlab Study</a><br><br><br><br>
+
  
 
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 +
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    <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Materials and Methods</b></h2>
 +
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 +
 +
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 +
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 +
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 +
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 +
    <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Reference</b></h2>
 +
    <hr style="width:50px;border:5px solid red" class="w3-round">
 +
 +
    <p style="font-size: 16px; text-indent:1em">
 +
    <a href=https://2015.igem.org/Team:Tokyo_Tech/Experiment/Interlab>2015 Tokyo_Tech InterLab</a>, accessed Augst 8, 2017
 +
    </p><br>
 +
 +
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Latest revision as of 00:17, 2 November 2017

<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

InterLab

Introduction

In iGEM 2017, the InterLab Study is one of the bronze medal criteria. We measured the GFP expression level of the six designated devices, positive control and negative control using the plate reader. It was the second time for our team to join this InterLab Study (The first time in iGEM 2015).


Summary

Our purpose was to investigate the RBS intensity and promoter intensity effects on gene expression by obtaining the fluorescence data of samples and comparing them. We prepared Device1〜Device6, Positive Control and Negative Control as shown below.


Fig. 1 Designated devices

Results

1. We obtained LUDOX correction values.


Fig. 2 LUDOX correct value

2. We obtained the two Fluorescein Standard Curve (normal and log scale).


Fig. 3 Fluorescein Standard Curve
Fig. 4 Fluorescein Standard Curve (log scale)

3. We obtained the graph bellow. The RFU of GFP / Turbidity was in the following order, Device1>Device4>Device2>Positive Control>Device5>Device3>Device6>Negative Control.


Fig. 5 Fluorescein (using AVERGE)
Fig. 6 Fluorescein (using GEOMEAN)

Discussion

Judging from the results, the gene expression is controlled by the intensity of promoter and the intensity of RBS. The promoter intensity of is supposed to be in the following order, J23101>J23106>J23117. The RBS intensity of I13504 is supposed to be higher than that of BCD2.E0040. Moreover, it is considered that the GFP expression level is dependent on individual to some extent.


Materials and Methods

Reference

2015 Tokyo_Tech InterLab, accessed Augst 8, 2017


Hajime Fujita with W3.CSS: All Rights Reserved