Difference between revisions of "Team:NUDT CHINA/InterLab"

 
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   <p style="font-size:36px;margin: 0 0%;padding: 5% 0 0 10%;color: white;">Designed Protein Degradation Method Based on</p>
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<p style="font-size:36px;margin: 0 0%;padding: 0 0 0 10%;color: white;">Trim21 And Nanobody&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;--&nbsp;InterLab</p>
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<div class="col-md-12 column" alt="cover_photo" style="width:100%;height:250px;background-size:cover;background-position:0px -350px; background-image:url(https://static.igem.org/mediawiki/2016/thumb/3/3f/NUDT_CHINA2016_FILES-img-banner-3.0.png/800px-NUDT_CHINA2016_FILES-img-banner-3.0.png);background-repeat:no-repeat;">
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   <p style="font-size:36px;margin: 0 0%;padding: 8% 0 0 10%;color: white;">Development of A Novel</p>
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<p style="font-size:36px;margin: 0 0%;padding: 0 0 0 10%;color: white;">Blood-MicroRNA Handy Detection System with CRISPR</p></div>
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      <h1 style="font-size: 40px;font-weight: 900">InterLab</h1>
 
      <h1 style="font-size: 40px;font-weight: 900">InterLab</h1>
 
                                       <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Introduction</h2>
 
                                       <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Introduction</h2>
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<p alt="content_add" style="font-size: 18px;">The InterLab this year has been updated to a more detailed protocol. During the InterLab study, we used the LUDOX, fluorescence and the plasmids that were shipped along with the distribution kit, and closely followed the InterLab study protocol. </p>
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<p alt="content_add" style="font-size: 18px;">The InterLab this year has been updated to a more detailed protocol. During the InterLab study, we used the LUDOX, fluorescence and the plasmids that were shipped along with the distribution kit, and closely followed the InterLab study protocol.We are really happy to receive the mail that our interlab study has beed accepted,it feels like a tiny victory!! </p>
 
<p alt="content_add" style="font-size: 18px;">
 
<p alt="content_add" style="font-size: 18px;">
 
Through the InterLab 2018 study, we experienced a lot in the measuring work, and surprisingly got to know more about the instruments that usually were ignored by us. Actually, it is the measuring instruments that help us complete the project every year!!  
 
Through the InterLab 2018 study, we experienced a lot in the measuring work, and surprisingly got to know more about the instruments that usually were ignored by us. Actually, it is the measuring instruments that help us complete the project every year!!  
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</div>
 
</div>
  
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                                 <div class="col-md-push-1 col-md-10">
 
                                 <div class="col-md-push-1 col-md-10">
 
                                       <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Methods and Design</h2>
 
                                       <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Methods and Design</h2>
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                                         <p alt="content_add" style="font-size: 18px;">Test Device6 (BBa_J364009) </p>
 
                                         <p alt="content_add" style="font-size: 18px;">Test Device6 (BBa_J364009) </p>
 
                                       <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2. Preparation</h3>
 
                                       <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2. Preparation</h3>
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<p alt="content_add" style="font-size: 18px;">To start with, our team transformed E.coli strain DH5α with the provided plasmids, namely Test Device 1,2,3,4,5,6, Positive Control, and Negative Control. As long as colonies had emerged on Cm+ Resistance Media, we picked up 2 colonies in each petrie dish and cultured them at 37℃ with 220 rpm frequency for 14 hours. When the bacteria solutions were turbid enough, we began the following process.
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<p alt="content_add" style="font-size: 18px;">To start with, our team transformed E.coli strain DH5α with the provided plasmids, namely Test Device 1,2,3,4,5,6, Positive Control, and Negative Control. As long as colonies had emerged on Cm<sup>+</sup> Resistance Media, we picked up 2 colonies in each petrie dish and cultured them at 37℃ with 220 rpm frequency for 14 hours. When the bacteria solutions were turbid enough, we began the following process.
 +
</p>
 +
  <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">3. OD600 Reference Point</h3>
 +
<p alt="content_add" style="font-size: 18px;">With plate reader, we measured Abs600 of the LUDOX and H2O. The H2O measurement served as the background. Both included 4 technical replicates to enhance the reliability of the results. Comparing to the standard OD600 reference given, which was 0.0425, we were able to achieve a ratio between OD600 and Abs600. The ratio was essential in converting Abs600 raw measurements into standard OD600 records.
 +
</p>
 +
  <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">4. Fluorescein Standard Curve</h3>
 +
<p alt="content_add" style="font-size: 18px;">Different concentrations of fluorescein were obtained by 2-fold serial dilution. In the first pipet, 200μL Fluorescein 1×stock solution was added. By removing half of the 200μL solution in previous pipets to later ones which already contained 100μL PBS, we were able to generate 11 solutions in half-descending fluorescence concentration. We also included another pipet with 100μL PBS only as blank. Within each concentration, we performed 4 replicates to calculate the more representative mean. After the results were recorded, all fluorescein concentrations were divided by average fluorescence measurement, of which the medium-high mean were calculated to diminish operation errors. This mean point would be used to set up the conversion between fluorescein concentration and fluorescence measurements in later steps.
 +
</p>
 +
  <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">5. OD600 and Fluorescence Measurements</h3>
 +
<p alt="content_add" style="font-size: 18px;">We tracked the original OD600 of the 16 samples so as to dilute them into 0.02. Once the original values were available, we integrated them into the dilution calculation sheet and followed the suggested volume to dilute each sample. After dilution, we confirmed that OD600 had reached exactly 0.02 or around. Our team set that time as T=0(h) and measured average OD600 from 4 replicates of each 16 samples to reduce technical error. Other 4 replicates were used for T=0(h) fluorescence measurements, with the same equipment and settings in Step 3. Henceforward, we recorded the OD600 and fluorescence when T=0 and T=6.
 +
</p>
 +
  <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">6. Equipments and Settings</h3>
 +
<p alt="content_add" style="font-size: 18px;">To obtain OD600 measurement, we employed Multiscan FC. Single wavelength of 600nm was used, and path length correction was turned off. To reduce measurement error, we also added a dynamic circulation of 5 times, with 2-second intervals.
 +
</p>
 +
<p alt="content_add" style="font-size: 18px;">The fluorescence measurement was obtained through Fluoroskan Ascent FL and Thermo Fisher. The filters used were 485nm and 538nm for excitation and emission respectively. The measurement cycled 5 times with intervals of 2 second.
 
</p>
 
</p>
 
</div>
 
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                                <div class="col-md-push-1 col-md-10">
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                                      <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Results & Discussion</h2>
 +
                                      <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">Calibration</h3>
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<p alt="content_add" style="font-size: 18px;">Calibration1: OD600 Reference point – LUDOX Protocol  </p>
 +
<p alt="content_add" style="font-size: 18px;">
 +
We used LUDOX CL-X (45% colloidal silica suspension) , ddH2O and 96 well plate(black with clear flat bottom preferred) to get the conversion factor, which can transform Abs600 measurements into comparable OD600 measurements.
 +
</p>
 +
<table class="table table-striped">
 +
<thead>
 +
<tr>
 +
  <th></th>
 +
  <th>LUDOX CL-X</th>
 +
  <th>H2O</th>
 +
</tr>
 +
  </thead>
 +
  <tbody>
 +
<tr>
 +
  <td>Replicate 1</td>
 +
  <td bgcolor="#00ccff" >
 +
                                          <p>0.062</p>
 +
                                          <td bgcolor="#00ccff" >
 +
  <p>0.039</p>
 +
</tr>
 +
<tr>
 +
  <td>Replicate 2</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.060</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.039</p>
 +
</tr>
 +
<tr>
 +
  <td>Replicate 3</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.066</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.038</p>
 +
</tr>
 +
<tr>
 +
  <td>Replicate 4</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.061</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.038</p>
 +
</tr>
 +
<tr>
 +
  <td>Arith. Mean</td>
 +
                                          <td bgcolor="#ffcc00">
 +
  <p>0.062</p>
 +
                                          <td bgcolor="#ffcc00">
 +
  <p>0.039</p>
 +
</tr>
 +
<tr>
 +
  <td>Corrected Abs600</td>
 +
                                          <td bgcolor="#ffcc00">
 +
  <p>0.024</p>
 +
  <td></td>
 +
</tr>
 +
<tr>
 +
  <td>Reference OD600</td>
 +
                                          <td bgcolor="#ffcc00">
 +
  <p>0.063</p>
 +
  <td></td>
 +
</tr>
 +
<tr>
 +
  <td>OD600/Abs600</td>
 +
                                          <td bgcolor="#ffcc00">
 +
  <p>2.654</p>
 +
  <td></td>
 +
</tr>
 +
  </tbody>
 +
</table> 
 +
                                      <h4 alt="photo_detail" style="text-align: center;">Fig.1 OD600 reference point</h4>
 +
 +
</div>
 +
 
 +
                                <div class="col-md-push-1 col-md-10">
 +
<p alt="content_add" style="font-size: 18px;">Calibration2: Particle Standard Curve – Microsphere Protocol  </p>
 +
<p alt="content_add" style="font-size: 18px;">
 +
Following the protocol, we prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. We got a particle standard curve and transferred it into log scale, which are shown as below.
 +
</p>
 +
</div>
 +
 
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/thumb/3/38/T--NUDT_CHINA--Particle.png/800px-T--NUDT_CHINA--Particle.png" />
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<p alt="photo_detail" style="text-align: center;">This is the sign of the famous game!</p>
+
<h4 alt="photo_detail" style="text-align: center;">Fig.2 Particle standard curve</h4>
 
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                                        <div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
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<!-- <p class="text-center text-info">this is the sign of igem</p> -->
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<img alt="800x433" src="https://static.igem.org/mediawiki/2018/5/5f/T--NUDT_CHINA--Particle_standard_curve_%28log_scale%29.png" />
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<p alt="photo_detail" style="text-align: center;"><em>This is the sign of the famous game!</em></p>
+
<h4 alt="photo_detail" style="text-align: center;">Fig.3 Particle standard curve (log scale)</h4>
 
</div>
 
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<div class="col-md-push-1 col-md-10">
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<p alt="content_add" style="font-size: 18px;">
 +
The values of the Particle standard curve are provides for convenience in a table.
 +
</p>
 
</div>
 
</div>
 +
<table class="table table-striped">
 +
<thead>
 +
<tr>
 +
  <th>Number of Particles</th>
 +
  <th>2.35E+08</th>
 +
  <th>1.18E+08</th>
 +
  <th>5.88E+07</th>
 +
  <th>2.94E+07</th>
 +
  <th>1.47E+07</th>
 +
  <th>7.35E+06</th>
 +
  <th>3.68E+06</th>
 +
  <th>1.84E+06</th>
 +
  <th>9.19E+05</th>
 +
  <th>4.60E+05</th>
 +
  <th>2.30E+05</th>
 +
  <th>0</th>
 +
</tr>
 +
  </thead>
 +
  <tbody>
 +
<tr>
 +
  <td>Replicate 1</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.668</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.393</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.201</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.122</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.074</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.056</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.046</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.043</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.038</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.036</p>
 +
</tr>
 +
<tr>
 +
  <td>Replicate 2</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.645</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.373</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.210</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.130</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.084</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.061</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.048</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.043</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.040</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.040</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.039</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
</tr>
 +
<tr>
 +
  <td>Replicate 3</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.619</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.349</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.211</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.129</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.084</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.060</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.049</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.044</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.038</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
</tr>
 +
<tr>
 +
  <td>Replicate 4</td>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.668</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.400</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.229</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.143</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.085</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.063</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.050</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.047</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.041</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.039</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.037</p>
 +
  <td bgcolor="#00ccff" >
 +
  <p>0.039</p>
 +
</tr>
 +
<tr>
 +
  <td>Arith.Mean</td>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.650</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.379</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.213</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.131</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.082</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.060</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.048</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.044</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.039</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.038</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.038</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.037</p>
 +
</tr>
 +
<tr>
 +
  <td>Arith.Std.Dev.</td>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.023</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.023</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.012</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.009</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.005</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.003</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.002</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.002</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.002</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.002</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.001</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.001</p>
 +
</tr>
 +
<tr>
 +
  <td>Arith.Net Mean</td>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.613</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.342</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.176</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.094</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.045</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.023</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.011</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.007</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.002</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.001</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p>0.000</p>
 +
  <td bgcolor="#ffcc00" >
 +
  <p></p>
 +
</tr>
 +
 +
  </tbody>
 +
</table> 
 +
                                      <h4 alt="photo_detail" style="text-align: center;">Fig.4 The values of Particle standard curve</h4>
 +
 +
</div>
 +
 
<div class="col-md-push-1 col-md-10">
 
<div class="col-md-push-1 col-md-10">
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<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
+
<p alt="content_add" style="font-size: 18px;">Calibration3: Fluorescence standard curve – Fluorescein Protocol </p>
 
<p alt="content_add" style="font-size: 18px;">
 
<p alt="content_add" style="font-size: 18px;">
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Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
We did a dilution series of fluorescein in four replicates. After recording the measurements, we generated a standard curve of fluorescence for fluorescein concentration and its log scale version, which are shown as below.
 
</p>
 
</p>
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<blockquote style="border-left-color: #18d26e;">
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<div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
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Maybe you want to write <strong>something interesting thing</strong> here!
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/6/68/T--NUDT_CHINA--Particle_standard_curve.png">
−
<small>Someone famous in <cite>Source Title</cite></small>
+
<h4 alt="photo_detail" style="text-align: center;">Fig.5 Particle standard curve</h4>
−
</blockquote>
+
</div>
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<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
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<div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
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<p alt="content_add" style="font-size: 18px;">
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/4/4a/T--NUDT_CHINA--Flourescein_standard_curve_%28log_scale%29.png">
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Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
<h4 alt="photo_detail" style="text-align: center;">Fig.6 Particle standard curve (log scale)</h4>
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</p>
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</div>
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<div class="col-md-12 column" alt = "main_content">
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<div class="col-md-6 col-md-push-3" alt="content_pictute">
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<div class="row center-block">
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<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
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<p alt="photo_detail" style="text-align: center;">This is the sign of the famous game!</p>
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</div>
+
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="col-md-push-1 col-md-10">
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<p alt="content_add" style="font-size: 18px;">hello everyone this is the another example-content part for more imformation. Hello everyone this is the another example-content part for more imformation.Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui. </p>
+
 
 +
<div class="col-md-push-1 col-md-10">
 +
 
<p alt="content_add" style="font-size: 18px;">
 
<p alt="content_add" style="font-size: 18px;">
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Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
We attached the details of the curves for reference.
 
</p>
 
</p>
−
<table class="table table-striped">
+
<table class="table table-striped">
−
  <caption>The example table for show some data</caption>
+
<thead>
−
  <thead>
+
 
<tr>
 
<tr>
−
  <th>name</th>
+
  <th>Fluorescein/a.u.</th>
−
  <th>city</th>
+
  <th></th>
−
  <th>mail</th>
+
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 
</tr>
 
</tr>
 
  </thead>
 
  </thead>
−
  <tbody>
+
<tbody>
 
<tr>
 
<tr>
−
  <td>Tanmay</td>
+
  <th>Fluorescein uM</th>
−
  <td>Bangalore</td>
+
  <th>10.00</th>
−
  <td>560001</td>
+
  <th>5.00</th>
 +
  <th>2.50</th>
 +
  <th>1.25</th>
 +
  <th>0.63</th>
 +
  <th>0.31</th>
 +
  <th>0.16</th>
 +
  <th>0.08</th>
 +
  <th>0.04</th>
 +
  <th>0.02</th>
 +
  <th>0.01</th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
  <td>Sachin</td>
+
  <th>uM fluorescein/a.u.:</th>
−
  <td>Mumbai</td>
+
  <th>4.38E-01</th>
−
  <td>400003</td>
+
  <th>3.69E-01</th>
 +
  <th>3.50E-01</th>
 +
  <th>3.35E-01</th>
 +
  <th>3.29E-01</th>
 +
  <th>3.37E-01</th>
 +
  <th>3.31E-01</th>
 +
  <th>3.39E-01</th>
 +
  <th>3.37E-01</th>
 +
  <th>3.51E-01</th>
 +
  <th>3.97E-01</th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
  <td>Uma</td>
+
  <th>Mean uM fluorescein/a.u.:</th>
−
  <td>Pune</td>
+
  <th></th>
−
  <td>411027</td>
+
  <th>3.44E-01</th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 
</tr>
 
</tr>
−
  </tbody>
+
<tr>
 +
  <th>MEFL/a.u.:</th>
 +
  <th></th>
 +
  <th>2.07E+12</th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
  <th></th>
 +
</tr>
 +
 
 +
</tbody>
 
</table>
 
</table>
−
<p alt="content_add" style="font-size: 18px;">
+
<h4 alt="photo_detail" style="text-align: center;">Fig.7 Detailed values of Fluorescein curve</h4>
−
Example block-level help text here.There are several variants of  derived from different organisms. In our project, we  . The structure of this protein has been determined with and without  The  a  lobe. The two lobes are positively charged towards the protein core to accommodate the negatively charged RNA. Each of these two lobes contains an RNase domain. At the  main is responsible for target cleavage. In contrast to other  two RNase domains of the NUC lobe are located at the outside of the protein, which is likely the reason collateral cleavage upon activation by binding to a matching target. These two domains have been labeled as red spots in Figure 2 and can be found at the interface between the green and pink domain.
+
</div>
 +
                                     
 +
 +
 
 +
 
 +
                                <div class="col-md-push-1 col-md-10">
 +
                                      <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">Cell measurement</h3>
 +
      <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2.1 Abs600</h3>
 +
                                      <p alt="content_add" style="font-size: 18px;">
 +
We have read two plates at the time point: 0 and 6 hours with the same conditions in our calibration measurements.
 
</p>
 
</p>
−
<p alt="content_add" style="font-size: 18px;">
+
                                      <p alt="content_add" style="font-size: 18px;">
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
We measured both fluorescence and absorbance of 2 biological replicates and 4 technical replicates. Abs600 values for device 5 are almost the lowest while the values of device 3 are almost the highest.
 +
</p>
 +
<table class="table table-striped">
 +
<caption>Abs600 Raw Readings:</caption>
 +
<thead>
 +
        <tr>
 +
        <th> </th>
 +
        <th> </th>
 +
        <th>Hour 0</th>
 +
        <th>Hour 6</th>
 +
        </tr>
 +
</thead>
 +
<tbody>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Neg. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.068</td>
 +
          <td>0.297</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.069</td>
 +
          <td>0.285</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.068</td>
 +
          <td>0.291</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.074</td>
 +
          <td>0.298</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Pos. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.072</td>
 +
          <td>0.296</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.071</td>
 +
          <td>0.288</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.075</td>
 +
          <td>0.284</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.068</td>
 +
          <td>0.285</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device1</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.068</td>
 +
          <td>0.239</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.22</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.073</td>
 +
          <td>0.223</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.069</td>
 +
          <td>0.224</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device2</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.066</td>
 +
          <td>0.268</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.071</td>
 +
          <td>0.27</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.068</td>
 +
          <td>0.268</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.066</td>
 +
          <td>0.273</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device3</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.069</td>
 +
          <td>0.425</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.358</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.072</td>
 +
          <td>0.343</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.074</td>
 +
          <td>0.347</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device4</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.067</td>
 +
          <td>0.21</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.217</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.067</td>
 +
          <td>0.217</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.07</td>
 +
          <td>0.221</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device5</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.066</td>
 +
          <td>0.161</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.155</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.069</td>
 +
          <td>0.16</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.066</td>
 +
          <td>0.164</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device6</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.067</td>
 +
          <td>0.294</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.068</td>
 +
          <td>0.284</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.067</td>
 +
          <td>0.286</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.071</td>
 +
          <td>0.285</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> LB+Chior(blank)</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.048</td>
 +
          <td>0.044</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.048</td>
 +
          <td>0.046</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.047</td>
 +
          <td>0.046</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.049</td>
 +
          <td>0.046</td>
 +
        </tr>
 +
</tbody>
 +
</table>
 +
                                      <h4 alt="photo_detail" style="text-align: center;">Fig.8 Raw Abs600 values(Colony 1)</h4>
 +
                                      <p alt="content_add" style="font-size: 18px;">
 +
We also used colony 2 series to do the same experiments. The features are similar to colony group. However, Abs600 value of device 2 reach the same level of device 3.
 
</p>
 
</p>
  
−
<div class="col-md-push-1 col-md-4 thumbnail">
+
<table class="table table-striped">
−
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
<caption>Abs600 Raw Readings:</caption>
−
</div>
+
<thead>
−
<div class="col-md-push-3 col-md-4 thumbnail">
+
        <tr>
−
<img alt="300x200" src="https://static.igem.org/mediawiki/2018/9/9b/2018_igem_menu_logo.svg" />
+
        <th> </th>
−
</div>
+
        <th> </th>
 +
        <th>Hour 0</th>
 +
        <th>Hour 6</th>
 +
        </tr>
 +
</thead>
 +
<tbody>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Neg. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.071</td>
 +
          <td>0.285</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.068</td>
 +
          <td>0.285</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.069</td>
 +
          <td>0.285</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.072</td>
 +
          <td>0.286</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Pos. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.068</td>
 +
          <td>0.299</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.296</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.071</td>
 +
          <td>0.297</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.071</td>
 +
          <td>0.303</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device1</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.068</td>
 +
          <td>0.238</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.249</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.073</td>
 +
          <td>0.244</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.069</td>
 +
          <td>0.246</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device2</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.065</td>
 +
          <td>0.295</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.064</td>
 +
          <td>0.299</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.069</td>
 +
          <td>0.301</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.07</td>
 +
          <td>0.306</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device3</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.068</td>
 +
          <td>0.295</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.068</td>
 +
          <td>0.304</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.069</td>
 +
          <td>0.298</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.067</td>
 +
          <td>0.302</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device4</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.069</td>
 +
          <td>0.217</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.075</td>
 +
          <td>0.22</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.068</td>
 +
          <td>0.218</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.067</td>
 +
          <td>0.217</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device5</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.066</td>
 +
          <td>0.156</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.16</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.07</td>
 +
          <td>0.162</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.068</td>
 +
          <td>0.161</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device6</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.064</td>
 +
          <td>0.268</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.067</td>
 +
          <td>0.281</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.065</td>
 +
          <td>0.269</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.069</td>
 +
          <td>0.272</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> LB+Chior(blank)</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.048</td>
 +
          <td>0.043</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.045</td>
 +
          <td>0.046</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.048</td>
 +
          <td>0.045</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.046</td>
 +
          <td>0.046</td>
 +
        </tr>
 +
</tbody>
 +
</table>
 +
                                      <h4 alt="photo_detail" style="text-align: center;">Fig.9 Raw Abs600 values(Colony 2)</h4>
 +
      <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2.2 Fluorescence</h3>
 +
                                      <p alt="content_add" style="font-size: 18px;">
 +
We used same colonies to measure their fluorescence values of device.
 +
</p>
 +
                                      <p alt="content_add" style="font-size: 18px;">
 +
As it is shown in figure 8, Fluorescence value of device 1 is the highest while the value of device 3 is the lowest, which approaches to the value of counterpart of negative control.
 +
</p>
 +
 
 +
<table class="table table-striped">
 +
<caption>Fluorescence Raw Readings:</caption>
 +
<thead>
 +
        <tr>
 +
        <th> </th>
 +
        <th> </th>
 +
        <th>Hour 0</th>
 +
        <th>Hour 6</th>
 +
        </tr>
 +
</thead>
 +
<tbody>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Neg. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.3566</td>
 +
          <td>0.3961</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.3743</td>
 +
          <td>0.4148</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.3855</td>
 +
          <td>0.4076</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.3704</td>
 +
          <td>0.4111</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Pos. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.5663</td>
 +
          <td>1.337</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5666</td>
 +
          <td>1.409</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5281</td>
 +
          <td>1.396</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5371</td>
 +
          <td>1.382</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device1</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.7175</td>
 +
          <td>2.538</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.7509</td>
 +
          <td>2.789</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.7584</td>
 +
          <td>2.656</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.724</td>
 +
          <td>2.656</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device2</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.6816</td>
 +
          <td>1.617</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.6791</td>
 +
          <td>1.735</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.6787</td>
 +
          <td>1.769</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.7568</td>
 +
          <td>1.776</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device3</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.3702</td>
 +
          <td>0.4131</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.3774</td>
 +
          <td>0.4373</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.373</td>
 +
          <td>0.4332</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.391</td>
 +
          <td>0.4299</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device4</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.5216</td>
 +
          <td>0.1.317</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5383</td>
 +
          <td>1.387</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5383</td>
 +
          <td>1.401</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.527</td>
 +
          <td>1.354</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device5</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.4981</td>
 +
          <td>1.145</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5158</td>
 +
          <td>1.228</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5069</td>
 +
          <td>1.198</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5063</td>
 +
          <td>1.194</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device6</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.4791</td>
 +
          <td>0.8039</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5</td>
 +
          <td>0.8388</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.482</td>
 +
          <td>0.8833</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5077</td>
 +
          <td>0.7948</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> LB+Chior(blank)</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.3488</td>
 +
          <td>0.3416</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.3577</td>
 +
          <td>0.3606</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.3647</td>
 +
          <td>0.36</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.3229</td>
 +
          <td>0.3624</td>
 +
        </tr>
 +
</tbody>
 +
</table>
 +
                                      <h4 alt="photo_detail" style="text-align: center;">Fig.10 Raw Fluorescence values(Colony 1)</h4>
 +
                                      <p alt="content_add" style="font-size: 18px;">
 +
At the second time, results of almost devices are in agreement to the first time, nevertheless, fluorescence value for device 2, which going to the highest.
 +
</p>
 +
 
 +
<table class="table table-striped">
 +
<caption>Fluorescence Raw Readings:</caption>
 +
<thead>
 +
        <tr>
 +
        <th> </th>
 +
        <th> </th>
 +
        <th>Hour 0</th>
 +
        <th>Hour 6</th>
 +
        </tr>
 +
</thead>
 +
<tbody>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Neg. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.3664</td>
 +
          <td>0.3724</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.3619</td>
 +
          <td>0.3871</td>
 +
        </tr>
 +
        <tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.3478</td>
 +
          <td>0.4008</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.3743</td>
 +
          <td>0.4208</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Pos. Control</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.5199</td>
 +
          <td>1.259</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5257</td>
 +
          <td>1.31</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5044</td>
 +
          <td>1.312</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5163</td>
 +
          <td>1.323</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device1</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.5493</td>
 +
          <td>1.791</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5729</td>
 +
          <td>1.941</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5377</td>
 +
          <td>1.89</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5425</td>
 +
          <td>1.937</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device2</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.629</td>
 +
          <td>2.095</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.615</td>
 +
          <td>2.142</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5999</td>
 +
          <td>2.266</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.6153</td>
 +
          <td>2.303</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device3</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.3725</td>
 +
          <td>0.4169</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.3825</td>
 +
          <td>0.4468</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.3815</td>
 +
          <td>0.4437</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.3767</td>
 +
          <td>0.4373</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device4</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.5816</td>
 +
          <td>1.26</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5933</td>
 +
          <td>1.299</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5749</td>
 +
          <td>1.39</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5997</td>
 +
          <td>1.344</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device5</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.5378</td>
 +
          <td>1.042</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.5532</td>
 +
          <td>0.1.093</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.5525</td>
 +
          <td>1.081</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.5725</td>
 +
          <td>1.092</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device6</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.4351</td>
 +
          <td>0.7296</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.4611</td>
 +
          <td>0.8149</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.4447</td>
 +
          <td>0.8102</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.4324</td>
 +
          <td>0.7783</td>
 +
        </tr>
 +
        <tr>
 +
          <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> LB+Chior(blank)</th>
 +
          <td> Replicate 1</td>
 +
          <td>0.3476</td>
 +
          <td>0.3708</td>
 +
        </tr>
 +
          <td>Replicate 2</td>
 +
          <td>0.3606</td>
 +
          <td>0.3631</td>
 +
        </tr>
 +
          <td>Replicate 3</td>
 +
          <td>0.347</td>
 +
          <td>0.3567</td>
 +
        </tr>
 +
          <td>Replicate 4</td>
 +
          <td>0.3223</td>
 +
          <td>0.3567</td>
 +
        </tr>
 +
</tbody>
 +
</table>
 +
                                      <h4 alt="photo_detail" style="text-align: center;">Fig.11 Raw Fluorescence values(Colony 2)</h4>
 
</div>
 
</div>
−
<div class="col-md-push-1 col-md-10">
+
 
−
<p alt="content_add" style="font-size: 18px;">
+
                                <div class="col-md-push-1 col-md-10">
−
Donec id elit non mi porta gravida at eget metus. Fusce dapibus, tellus ac cursus commodo, tortor mauris condimentum nibh, ut fermentum massa justo sit amet risus. Etiam porta sem malesuada magna mollis euismod. Donec sed odio dui.
+
                                      <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">Protocol:</h3>
 +
                                      <p alt="content_add" style="font-size: 18px;">
 +
We measured the Abs600 of our cell cultures and then used the appropriate ratio between Abs600 and OD600 to calculate the OD600 value. After getting the results, we diluted our overnight culture to OD600=1.0 for each culture and checked it. The data of Fluorescence per OD and Fluorescence per Particle at 0 and 6 hours is shown as below.
 +
</p>
 +
<table class="table table-striped">
 +
<thead>
 +
<tr>
 +
  <th>OD600/Abs600</th>
 +
  <th>2.65</th>
 +
</tr>
 +
<tr>
 +
  <th>uM Fluorescein/a.u.</th>
 +
  <th>0.344</th>
 +
</tr>
 +
<tr>
 +
  <th>uM Fluorescein/OD</th>
 +
</tr>
 +
  </thead>
 +
  <tbody>
 +
<tr>
 +
  <td>Hour 0:</td>
 +
  <td>Neg.Control</td>
 +
  <td>Pos.Control</td>
 +
  <td>Device1</td>
 +
  <td>Device2</td>
 +
  <td>Device3</td>
 +
  <td>Device4</td>
 +
  <td>Device5</td>
 +
  <td>Device6</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 1</td>
 +
  <td>1.410</td>
 +
  <td>1.175</td>
 +
  <td>2.391</td>
 +
  <td>2.398</td>
 +
  <td>0.132</td>
 +
  <td>1.180</td>
 +
  <td>1.076</td>
 +
  <td>0.889</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 2</td>
 +
  <td>1.290</td>
 +
  <td>1.178</td>
 +
  <td>2.684</td>
 +
  <td>1.812</td>
 +
  <td>0.134</td>
 +
  <td>1.233</td>
 +
  <td>1.079</td>
 +
  <td>0.923</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 3</td>
 +
  <td>1.009</td>
 +
  <td>0.757</td>
 +
  <td>1.964</td>
 +
  <td>1.939</td>
 +
  <td>0.043</td>
 +
  <td>1.126</td>
 +
  <td>0.838</td>
 +
  <td>0.761</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 4</td>
 +
  <td>1.111</td>
 +
  <td>1.462</td>
 +
  <td>2.601</td>
 +
  <td>3.310</td>
 +
  <td>0.353</td>
 +
  <td>1.261</td>
 +
  <td>1.399</td>
 +
  <td>1.089</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 1</td>
 +
  <td>0.972</td>
 +
  <td>1.117</td>
 +
  <td>1.869</td>
 +
  <td>2.147</td>
 +
  <td>0.161</td>
 +
  <td>1.445</td>
 +
  <td>1.370</td>
 +
  <td>0.709</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 2</td>
 +
  <td>0.931</td>
 +
  <td>0.973</td>
 +
  <td>1.530</td>
 +
  <td>1.737</td>
 +
  <td>0.123</td>
 +
  <td>1.006</td>
 +
  <td>1.135</td>
 +
  <td>0.592</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 3</td>
 +
  <td>0.972</td>
 +
  <td>0.888</td>
 +
  <td>1.302</td>
 +
  <td>1.562</td>
 +
  <td>0.213</td>
 +
  <td>1.478</td>
 +
  <td>1.211</td>
 +
  <td>0.745</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 4</td>
 +
  <td>0.986</td>
 +
  <td>1.006</td>
 +
  <td>1.503</td>
 +
  <td>1.583</td>
 +
  <td>0.336</td>
 +
  <td>1.713</td>
 +
  <td>1.475</td>
 +
  <td>0.621</td>
 +
</tr>
 +
<tr>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
</tr>
 +
<tr>
 +
  <td>Hour 6:</td>
 +
  <td>Neg.Control</td>
 +
  <td>Pos.Control</td>
 +
  <td>Device1</td>
 +
  <td>Device2</td>
 +
  <td>Device3</td>
 +
  <td>Device4</td>
 +
  <td>Device5</td>
 +
  <td>Device6</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 1</td>
 +
  <td>0.028</td>
 +
  <td>0.512</td>
 +
  <td>1.461</td>
 +
  <td>0.738</td>
 +
  <td>0.024</td>
 +
  <td>0.762</td>
 +
  <td>0.891</td>
 +
  <td>0.240</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 2</td>
 +
  <td>0.029</td>
 +
  <td>0.562</td>
 +
  <td>1.810</td>
 +
  <td>0.796</td>
 +
  <td>0.032</td>
 +
  <td>0.778</td>
 +
  <td>1.032</td>
 +
  <td>0.261</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 3</td>
 +
  <td>0.025</td>
 +
  <td>0.565</td>
 +
  <td>1.682</td>
 +
  <td>0.823</td>
 +
  <td>0.032</td>
 +
  <td>0.790</td>
 +
  <td>0.953</td>
 +
  <td>0.283</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 4</td>
 +
  <td>0.025</td>
 +
  <td>0.553</td>
 +
  <td>1.671</td>
 +
  <td>0.808</td>
 +
  <td>0.029</td>
 +
  <td>0.735</td>
 +
  <td>0.914</td>
 +
  <td>0.235</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 1</td>
 +
  <td>0.001</td>
 +
  <td>0.450</td>
 +
  <td>0.945</td>
 +
  <td>0.887</td>
 +
  <td>0.024</td>
 +
  <td>0.663</td>
 +
  <td>0.770</td>
 +
  <td>0.207</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 2</td>
 +
  <td>0.013</td>
 +
  <td>0.491</td>
 +
  <td>1.008</td>
 +
  <td>0.912</td>
 +
  <td>0.042</td>
 +
  <td>0.698</td>
 +
  <td>0.830</td>
 +
  <td>0.249</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 3</td>
 +
  <td>0.024</td>
 +
  <td>0.492</td>
 +
  <td>0.999</td>
 +
  <td>0.967</td>
 +
  <td>0.045</td>
 +
  <td>0.775</td>
 +
  <td>0.803</td>
 +
  <td>0.263</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 4</td>
 +
  <td>0.035</td>
 +
  <td>0.488</td>
 +
  <td>1.025</td>
 +
  <td>0.971</td>
 +
  <td>0.041</td>
 +
  <td>0.749</td>
 +
  <td>0.829</td>
 +
  <td>0.242</td>
 +
</tr>
 +
  </tbody>
 +
</table> 
 +
<h4 alt="photo_detail" style="text-align: center;">Fig.12 Fluorescence per OD</h4>
 +
 
 +
<table class="table table-striped">
 +
<thead>
 +
<tr>
 +
  <th>Particles/Abs600</th>
 +
  <th>3.29E+08</th>
 +
</tr>
 +
<tr>
 +
  <th>MEFL/a.u.</th>
 +
  <th>2.07E+12</th>
 +
</tr>
 +
<tr>
 +
  <th>MEFL/particle</th>
 +
</tr>
 +
  </thead>
 +
  <tbody>
 +
<tr>
 +
  <td>Hour 0:</td>
 +
  <td>Neg.Control</td>
 +
  <td>Pos.Control</td>
 +
  <td>Device1</td>
 +
  <td>Device2</td>
 +
  <td>Device3</td>
 +
  <td>Device4</td>
 +
  <td>Device5</td>
 +
  <td>Device6</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 1</td>
 +
  <td>6.84E+04</td>
 +
  <td>5.70E+04</td>
 +
  <td>1.16E+05</td>
 +
  <td>1.16E+05</td>
 +
  <td>6.41E+03</td>
 +
  <td>5.72E+04</td>
 +
  <td>5.22E+04</td>
 +
  <td>4.32E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 2</td>
 +
  <td>6.26E+04</td>
 +
  <td>5.71E+04</td>
 +
  <td>1.30E+05</td>
 +
  <td>8.79E+04</td>
 +
  <td>6.52E+03</td>
 +
  <td>5.98E+04</td>
 +
  <td>5.24E+04</td>
 +
  <td>4.48E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 3</td>
 +
  <td>4.90E+04</td>
 +
  <td>3.67E+04</td>
 +
  <td>9.53E+04</td>
 +
  <td>9.41E+04</td>
 +
  <td>2.09E+03</td>
 +
  <td>5.46E+04</td>
 +
  <td>4.07E+04</td>
 +
  <td>3.69E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 4</td>
 +
  <td>5.39E+04</td>
 +
  <td>7.09E+04</td>
 +
  <td>1.26E+05</td>
 +
  <td>1.61E+05</td>
 +
  <td>1.71E+04</td>
 +
  <td>6.12E+04</td>
 +
  <td>6.79E+04</td>
 +
  <td>5.29E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 1</td>
 +
  <td>4.71E+04</td>
 +
  <td>5.42E+04</td>
 +
  <td>9.07E+04</td>
 +
  <td>1.04E+05</td>
 +
  <td>7.83E+03</td>
 +
  <td>7.01E+04</td>
 +
  <td>6.65E+04</td>
 +
  <td>3.44E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 2</td>
 +
  <td>4.52E+04</td>
 +
  <td>4.72E+04</td>
 +
  <td>7.42E+04</td>
 +
  <td>8.42E+04</td>
 +
  <td>5.99E+03</td>
 +
  <td>4.88E+04</td>
 +
  <td>5.51E+04</td>
 +
  <td>2.87E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 3</td>
 +
  <td>4.72E+04</td>
 +
  <td>4.31E+04</td>
 +
  <td>6.32E+04</td>
 +
  <td>7.58E+04</td>
 +
  <td>1.03E+04</td>
 +
  <td>7.17E+04</td>
 +
  <td>5.88E+04</td>
 +
  <td>3.62E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 4</td>
 +
  <td>4.69E+04</td>
 +
  <td>4.88E+04</td>
 +
  <td>7.29E+04</td>
 +
  <td>7.68E+04</td>
 +
  <td>1.63E+04</td>
 +
  <td>8.31E+04</td>
 +
  <td>7.16E+04</td>
 +
  <td>3.01E+04</td>
 +
</tr>
 +
<tr>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
  <td></td>
 +
</tr>
 +
<tr>
 +
  <td>Hour 6:</td>
 +
  <td>Neg.Control</td>
 +
  <td>Pos.Control</td>
 +
  <td>Device1</td>
 +
  <td>Device2</td>
 +
  <td>Device3</td>
 +
  <td>Device4</td>
 +
  <td>Device5</td>
 +
  <td>Device6</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 1</td>
 +
  <td>1.36E+03</td>
 +
  <td>2.49E+04</td>
 +
  <td>7.09E+04</td>
 +
  <td>3.58E+04</td>
 +
  <td>1.18E+03</td>
 +
  <td>3.70E+04</td>
 +
  <td>4.32E+04</td>
 +
  <td>1.16E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 2</td>
 +
  <td>1.43E+03</td>
 +
  <td>2.73E+04</td>
 +
  <td>8.78E+04</td>
 +
  <td>3.86E+04</td>
 +
  <td>1.55E+03</td>
 +
  <td>3.78E+04</td>
 +
  <td>5.01E+04</td>
 +
  <td>1.26E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 3</td>
 +
  <td>1.22E+03</td>
 +
  <td>2.74E+04</td>
 +
  <td>8.16E+04</td>
 +
  <td>3.99E+04</td>
 +
  <td>1.55E+03</td>
 +
  <td>3.83E+04</td>
 +
  <td>4.63E+04</td>
 +
  <td>1.37E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 1,Replicate 4</td>
 +
  <td>1.22E+03</td>
 +
  <td>2.68E+04</td>
 +
  <td>8.11E+04</td>
 +
  <td>3.92E+04</td>
 +
  <td>1.41E+03</td>
 +
  <td>3.57E+04</td>
 +
  <td>4.43E+04</td>
 +
  <td>1.14E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 1</td>
 +
  <td>4.16E+01</td>
 +
  <td>2.18E+04</td>
 +
  <td>4.58E+04</td>
 +
  <td>4.31E+04</td>
 +
  <td>1.15E+03</td>
 +
  <td>3.22E+04</td>
 +
  <td>3.74E+04</td>
 +
  <td>1.00E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 2</td>
 +
  <td>6.32E+02</td>
 +
  <td>2.38E+04</td>
 +
  <td>4.89E+04</td>
 +
  <td>4.42E+04</td>
 +
  <td>2.04E+03</td>
 +
  <td>3.38E+04</td>
 +
  <td>4.03E+04</td>
 +
  <td>1.21E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 3</td>
 +
  <td>1.16E+03</td>
 +
  <td>2.39E+04</td>
 +
  <td>4.85E+04</td>
 +
  <td>4.69E+04</td>
 +
  <td>2.16E+03</td>
 +
  <td>3.76E+04</td>
 +
  <td>3.90E+04</td>
 +
  <td>1.27E+04</td>
 +
</tr>
 +
<tr>
 +
  <td>Colony 2,Replicate 4</td>
 +
  <td>1.68E+03</td>
 +
  <td>2.37E+04</td>
 +
  <td>4.97E+04</td>
 +
  <td>4.71E+04</td>
 +
  <td>1.98E+03</td>
 +
  <td>3.63E+04</td>
 +
  <td>4.02E+04</td>
 +
  <td>1.17E+04</td>
 +
</tr>
 +
  </tbody>
 +
</table> 
 +
<h4 alt="photo_detail" style="text-align: center;">Fig.13 Fluorescence per Particle</h4>
 +
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Finally, we counted the colonies on each plate with fewer than 300 colonies. Then, we multiplied by the Final Dilution Factor: 8  . We recorded the CFU per 1mL of each OD600 = 0.1 culture.
 
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There are several variants of  derived from different organisms. In our project, we  . The structure of this protein has been determined with and without  The  a  lobe. The two lobes are positively charged towards the protein core to accommodate the negatively charged RNA. Each of these two lobes contains an RNase domain. At the  main is responsible for target cleavage. In contrast to other  two RNase domains of the NUC lobe are located at the outside of the protein, which is likely the reason collateral cleavage upon activation by binding to a matching target. These two domains have been labeled as red spots in Figure 2 and can be found at the interface between the green and pink domain.
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Latest revision as of 01:55, 18 October 2018

Designed Protein Degradation Method Based on

Trim21 And Nanobody              -- InterLab

InterLab

Introduction

The InterLab this year has been updated to a more detailed protocol. During the InterLab study, we used the LUDOX, fluorescence and the plasmids that were shipped along with the distribution kit, and closely followed the InterLab study protocol.We are really happy to receive the mail that our interlab study has beed accepted,it feels like a tiny victory!!

Through the InterLab 2018 study, we experienced a lot in the measuring work, and surprisingly got to know more about the instruments that usually were ignored by us. Actually, it is the measuring instruments that help us complete the project every year!!

Methods and Design

1. InterLab Parts

Positive Control (BBa_I20270)

NegativeControl (BBa_R0040)

Test Device1 (BBa_J364000)

Test Device2 (BBa_J364001)

Test Device3 (BBa_J364002)

Test Device4 (BBa_J364007)

Test Device5 (BBa_J364008)

Test Device6 (BBa_J364009)

2. Preparation

To start with, our team transformed E.coli strain DH5α with the provided plasmids, namely Test Device 1,2,3,4,5,6, Positive Control, and Negative Control. As long as colonies had emerged on Cm+ Resistance Media, we picked up 2 colonies in each petrie dish and cultured them at 37℃ with 220 rpm frequency for 14 hours. When the bacteria solutions were turbid enough, we began the following process.

3. OD600 Reference Point

With plate reader, we measured Abs600 of the LUDOX and H2O. The H2O measurement served as the background. Both included 4 technical replicates to enhance the reliability of the results. Comparing to the standard OD600 reference given, which was 0.0425, we were able to achieve a ratio between OD600 and Abs600. The ratio was essential in converting Abs600 raw measurements into standard OD600 records.

4. Fluorescein Standard Curve

Different concentrations of fluorescein were obtained by 2-fold serial dilution. In the first pipet, 200μL Fluorescein 1×stock solution was added. By removing half of the 200μL solution in previous pipets to later ones which already contained 100μL PBS, we were able to generate 11 solutions in half-descending fluorescence concentration. We also included another pipet with 100μL PBS only as blank. Within each concentration, we performed 4 replicates to calculate the more representative mean. After the results were recorded, all fluorescein concentrations were divided by average fluorescence measurement, of which the medium-high mean were calculated to diminish operation errors. This mean point would be used to set up the conversion between fluorescein concentration and fluorescence measurements in later steps.

5. OD600 and Fluorescence Measurements

We tracked the original OD600 of the 16 samples so as to dilute them into 0.02. Once the original values were available, we integrated them into the dilution calculation sheet and followed the suggested volume to dilute each sample. After dilution, we confirmed that OD600 had reached exactly 0.02 or around. Our team set that time as T=0(h) and measured average OD600 from 4 replicates of each 16 samples to reduce technical error. Other 4 replicates were used for T=0(h) fluorescence measurements, with the same equipment and settings in Step 3. Henceforward, we recorded the OD600 and fluorescence when T=0 and T=6.

6. Equipments and Settings

To obtain OD600 measurement, we employed Multiscan FC. Single wavelength of 600nm was used, and path length correction was turned off. To reduce measurement error, we also added a dynamic circulation of 5 times, with 2-second intervals.

The fluorescence measurement was obtained through Fluoroskan Ascent FL and Thermo Fisher. The filters used were 485nm and 538nm for excitation and emission respectively. The measurement cycled 5 times with intervals of 2 second.

Results & Discussion

Calibration

Calibration1: OD600 Reference point – LUDOX Protocol

We used LUDOX CL-X (45% colloidal silica suspension) , ddH2O and 96 well plate(black with clear flat bottom preferred) to get the conversion factor, which can transform Abs600 measurements into comparable OD600 measurements.

LUDOX CL-X H2O
Replicate 1

0.062

0.039

Replicate 2

0.060

0.039

Replicate 3

0.066

0.038

Replicate 4

0.061

0.038

Arith. Mean

0.062

0.039

Corrected Abs600

0.024
Reference OD600

0.063
OD600/Abs600

2.654

Fig.1 OD600 reference point

Calibration2: Particle Standard Curve – Microsphere Protocol

Following the protocol, we prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. We got a particle standard curve and transferred it into log scale, which are shown as below.

300x200

Fig.2 Particle standard curve

800x433

Fig.3 Particle standard curve (log scale)

The values of the Particle standard curve are provides for convenience in a table.

Number of Particles 2.35E+08 1.18E+08 5.88E+07 2.94E+07 1.47E+07 7.35E+06 3.68E+06 1.84E+06 9.19E+05 4.60E+05 2.30E+05 0
Replicate 1

0.668

0.393

0.201

0.122

0.074

0.056

0.046

0.043

0.038

0.037

0.037

0.036

Replicate 2

0.645

0.373

0.210

0.130

0.084

0.061

0.048

0.043

0.040

0.040

0.039

0.037

Replicate 3

0.619

0.349

0.211

0.129

0.084

0.060

0.049

0.044

0.038

0.037

0.037

0.037

Replicate 4

0.668

0.400

0.229

0.143

0.085

0.063

0.050

0.047

0.041

0.039

0.037

0.039

Arith.Mean

0.650

0.379

0.213

0.131

0.082

0.060

0.048

0.044

0.039

0.038

0.038

0.037

Arith.Std.Dev.

0.023

0.023

0.012

0.009

0.005

0.003

0.002

0.002

0.002

0.002

0.001

0.001

Arith.Net Mean

0.613

0.342

0.176

0.094

0.045

0.023

0.011

0.007

0.002

0.001

0.000

Fig.4 The values of Particle standard curve

Calibration3: Fluorescence standard curve – Fluorescein Protocol

We did a dilution series of fluorescein in four replicates. After recording the measurements, we generated a standard curve of fluorescence for fluorescein concentration and its log scale version, which are shown as below.

300x200

Fig.5 Particle standard curve

300x200

Fig.6 Particle standard curve (log scale)

We attached the details of the curves for reference.

Fluorescein/a.u.
Fluorescein uM 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.02 0.01
uM fluorescein/a.u.: 4.38E-01 3.69E-01 3.50E-01 3.35E-01 3.29E-01 3.37E-01 3.31E-01 3.39E-01 3.37E-01 3.51E-01 3.97E-01
Mean uM fluorescein/a.u.: 3.44E-01
MEFL/a.u.: 2.07E+12

Fig.7 Detailed values of Fluorescein curve

Cell measurement

2.1 Abs600

We have read two plates at the time point: 0 and 6 hours with the same conditions in our calibration measurements.

We measured both fluorescence and absorbance of 2 biological replicates and 4 technical replicates. Abs600 values for device 5 are almost the lowest while the values of device 3 are almost the highest.

Abs600 Raw Readings:
Hour 0 Hour 6
Neg. Control Replicate 1 0.068 0.297
Replicate 2 0.069 0.285
Replicate 3 0.068 0.291
Replicate 4 0.074 0.298
Pos. Control Replicate 1 0.072 0.296
Replicate 2 0.071 0.288
Replicate 3 0.075 0.284
Replicate 4 0.068 0.285
Device1 Replicate 1 0.068 0.239
Replicate 2 0.067 0.22
Replicate 3 0.073 0.223
Replicate 4 0.069 0.224
Device2 Replicate 1 0.066 0.268
Replicate 2 0.071 0.27
Replicate 3 0.068 0.268
Replicate 4 0.066 0.273
Device3 Replicate 1 0.069 0.425
Replicate 2 0.067 0.358
Replicate 3 0.072 0.343
Replicate 4 0.074 0.347
Device4 Replicate 1 0.067 0.21
Replicate 2 0.067 0.217
Replicate 3 0.067 0.217
Replicate 4 0.07 0.221
Device5 Replicate 1 0.066 0.161
Replicate 2 0.067 0.155
Replicate 3 0.069 0.16
Replicate 4 0.066 0.164
Device6 Replicate 1 0.067 0.294
Replicate 2 0.068 0.284
Replicate 3 0.067 0.286
Replicate 4 0.071 0.285
LB+Chior(blank) Replicate 1 0.048 0.044
Replicate 2 0.048 0.046
Replicate 3 0.047 0.046
Replicate 4 0.049 0.046

Fig.8 Raw Abs600 values(Colony 1)

We also used colony 2 series to do the same experiments. The features are similar to colony group. However, Abs600 value of device 2 reach the same level of device 3.

Abs600 Raw Readings:
Hour 0 Hour 6
Neg. Control Replicate 1 0.071 0.285
Replicate 2 0.068 0.285
Replicate 3 0.069 0.285
Replicate 4 0.072 0.286
Pos. Control Replicate 1 0.068 0.299
Replicate 2 0.067 0.296
Replicate 3 0.071 0.297
Replicate 4 0.071 0.303
Device1 Replicate 1 0.068 0.238
Replicate 2 0.067 0.249
Replicate 3 0.073 0.244
Replicate 4 0.069 0.246
Device2 Replicate 1 0.065 0.295
Replicate 2 0.064 0.299
Replicate 3 0.069 0.301
Replicate 4 0.07 0.306
Device3 Replicate 1 0.068 0.295
Replicate 2 0.068 0.304
Replicate 3 0.069 0.298
Replicate 4 0.067 0.302
Device4 Replicate 1 0.069 0.217
Replicate 2 0.075 0.22
Replicate 3 0.068 0.218
Replicate 4 0.067 0.217
Device5 Replicate 1 0.066 0.156
Replicate 2 0.067 0.16
Replicate 3 0.07 0.162
Replicate 4 0.068 0.161
Device6 Replicate 1 0.064 0.268
Replicate 2 0.067 0.281
Replicate 3 0.065 0.269
Replicate 4 0.069 0.272
LB+Chior(blank) Replicate 1 0.048 0.043
Replicate 2 0.045 0.046
Replicate 3 0.048 0.045
Replicate 4 0.046 0.046

Fig.9 Raw Abs600 values(Colony 2)

2.2 Fluorescence

We used same colonies to measure their fluorescence values of device.

As it is shown in figure 8, Fluorescence value of device 1 is the highest while the value of device 3 is the lowest, which approaches to the value of counterpart of negative control.

Fluorescence Raw Readings:
Hour 0 Hour 6
Neg. Control Replicate 1 0.3566 0.3961
Replicate 2 0.3743 0.4148
Replicate 3 0.3855 0.4076
Replicate 4 0.3704 0.4111
Pos. Control Replicate 1 0.5663 1.337
Replicate 2 0.5666 1.409
Replicate 3 0.5281 1.396
Replicate 4 0.5371 1.382
Device1 Replicate 1 0.7175 2.538
Replicate 2 0.7509 2.789
Replicate 3 0.7584 2.656
Replicate 4 0.724 2.656
Device2 Replicate 1 0.6816 1.617
Replicate 2 0.6791 1.735
Replicate 3 0.6787 1.769
Replicate 4 0.7568 1.776
Device3 Replicate 1 0.3702 0.4131
Replicate 2 0.3774 0.4373
Replicate 3 0.373 0.4332
Replicate 4 0.391 0.4299
Device4 Replicate 1 0.5216 0.1.317
Replicate 2 0.5383 1.387
Replicate 3 0.5383 1.401
Replicate 4 0.527 1.354
Device5 Replicate 1 0.4981 1.145
Replicate 2 0.5158 1.228
Replicate 3 0.5069 1.198
Replicate 4 0.5063 1.194
Device6 Replicate 1 0.4791 0.8039
Replicate 2 0.5 0.8388
Replicate 3 0.482 0.8833
Replicate 4 0.5077 0.7948
LB+Chior(blank) Replicate 1 0.3488 0.3416
Replicate 2 0.3577 0.3606
Replicate 3 0.3647 0.36
Replicate 4 0.3229 0.3624

Fig.10 Raw Fluorescence values(Colony 1)

At the second time, results of almost devices are in agreement to the first time, nevertheless, fluorescence value for device 2, which going to the highest.

Fluorescence Raw Readings:
Hour 0 Hour 6
Neg. Control Replicate 1 0.3664 0.3724
Replicate 2 0.3619 0.3871
Replicate 3 0.3478 0.4008
Replicate 4 0.3743 0.4208
Pos. Control Replicate 1 0.5199 1.259
Replicate 2 0.5257 1.31
Replicate 3 0.5044 1.312
Replicate 4 0.5163 1.323
Device1 Replicate 1 0.5493 1.791
Replicate 2 0.5729 1.941
Replicate 3 0.5377 1.89
Replicate 4 0.5425 1.937
Device2 Replicate 1 0.629 2.095
Replicate 2 0.615 2.142
Replicate 3 0.5999 2.266
Replicate 4 0.6153 2.303
Device3 Replicate 1 0.3725 0.4169
Replicate 2 0.3825 0.4468
Replicate 3 0.3815 0.4437
Replicate 4 0.3767 0.4373
Device4 Replicate 1 0.5816 1.26
Replicate 2 0.5933 1.299
Replicate 3 0.5749 1.39
Replicate 4 0.5997 1.344
Device5 Replicate 1 0.5378 1.042
Replicate 2 0.5532 0.1.093
Replicate 3 0.5525 1.081
Replicate 4 0.5725 1.092
Device6 Replicate 1 0.4351 0.7296
Replicate 2 0.4611 0.8149
Replicate 3 0.4447 0.8102
Replicate 4 0.4324 0.7783
LB+Chior(blank) Replicate 1 0.3476 0.3708
Replicate 2 0.3606 0.3631
Replicate 3 0.347 0.3567
Replicate 4 0.3223 0.3567

Fig.11 Raw Fluorescence values(Colony 2)

Protocol:

We measured the Abs600 of our cell cultures and then used the appropriate ratio between Abs600 and OD600 to calculate the OD600 value. After getting the results, we diluted our overnight culture to OD600=1.0 for each culture and checked it. The data of Fluorescence per OD and Fluorescence per Particle at 0 and 6 hours is shown as below.

OD600/Abs600 2.65
uM Fluorescein/a.u. 0.344
uM Fluorescein/OD
Hour 0: Neg.Control Pos.Control Device1 Device2 Device3 Device4 Device5 Device6
Colony 1,Replicate 1 1.410 1.175 2.391 2.398 0.132 1.180 1.076 0.889
Colony 1,Replicate 2 1.290 1.178 2.684 1.812 0.134 1.233 1.079 0.923
Colony 1,Replicate 3 1.009 0.757 1.964 1.939 0.043 1.126 0.838 0.761
Colony 1,Replicate 4 1.111 1.462 2.601 3.310 0.353 1.261 1.399 1.089
Colony 2,Replicate 1 0.972 1.117 1.869 2.147 0.161 1.445 1.370 0.709
Colony 2,Replicate 2 0.931 0.973 1.530 1.737 0.123 1.006 1.135 0.592
Colony 2,Replicate 3 0.972 0.888 1.302 1.562 0.213 1.478 1.211 0.745
Colony 2,Replicate 4 0.986 1.006 1.503 1.583 0.336 1.713 1.475 0.621
Hour 6: Neg.Control Pos.Control Device1 Device2 Device3 Device4 Device5 Device6
Colony 1,Replicate 1 0.028 0.512 1.461 0.738 0.024 0.762 0.891 0.240
Colony 1,Replicate 2 0.029 0.562 1.810 0.796 0.032 0.778 1.032 0.261
Colony 1,Replicate 3 0.025 0.565 1.682 0.823 0.032 0.790 0.953 0.283
Colony 1,Replicate 4 0.025 0.553 1.671 0.808 0.029 0.735 0.914 0.235
Colony 2,Replicate 1 0.001 0.450 0.945 0.887 0.024 0.663 0.770 0.207
Colony 2,Replicate 2 0.013 0.491 1.008 0.912 0.042 0.698 0.830 0.249
Colony 2,Replicate 3 0.024 0.492 0.999 0.967 0.045 0.775 0.803 0.263
Colony 2,Replicate 4 0.035 0.488 1.025 0.971 0.041 0.749 0.829 0.242

Fig.12 Fluorescence per OD

Particles/Abs600 3.29E+08
MEFL/a.u. 2.07E+12
MEFL/particle
Hour 0: Neg.Control Pos.Control Device1 Device2 Device3 Device4 Device5 Device6
Colony 1,Replicate 1 6.84E+04 5.70E+04 1.16E+05 1.16E+05 6.41E+03 5.72E+04 5.22E+04 4.32E+04
Colony 1,Replicate 2 6.26E+04 5.71E+04 1.30E+05 8.79E+04 6.52E+03 5.98E+04 5.24E+04 4.48E+04
Colony 1,Replicate 3 4.90E+04 3.67E+04 9.53E+04 9.41E+04 2.09E+03 5.46E+04 4.07E+04 3.69E+04
Colony 1,Replicate 4 5.39E+04 7.09E+04 1.26E+05 1.61E+05 1.71E+04 6.12E+04 6.79E+04 5.29E+04
Colony 2,Replicate 1 4.71E+04 5.42E+04 9.07E+04 1.04E+05 7.83E+03 7.01E+04 6.65E+04 3.44E+04
Colony 2,Replicate 2 4.52E+04 4.72E+04 7.42E+04 8.42E+04 5.99E+03 4.88E+04 5.51E+04 2.87E+04
Colony 2,Replicate 3 4.72E+04 4.31E+04 6.32E+04 7.58E+04 1.03E+04 7.17E+04 5.88E+04 3.62E+04
Colony 2,Replicate 4 4.69E+04 4.88E+04 7.29E+04 7.68E+04 1.63E+04 8.31E+04 7.16E+04 3.01E+04
Hour 6: Neg.Control Pos.Control Device1 Device2 Device3 Device4 Device5 Device6
Colony 1,Replicate 1 1.36E+03 2.49E+04 7.09E+04 3.58E+04 1.18E+03 3.70E+04 4.32E+04 1.16E+04
Colony 1,Replicate 2 1.43E+03 2.73E+04 8.78E+04 3.86E+04 1.55E+03 3.78E+04 5.01E+04 1.26E+04
Colony 1,Replicate 3 1.22E+03 2.74E+04 8.16E+04 3.99E+04 1.55E+03 3.83E+04 4.63E+04 1.37E+04
Colony 1,Replicate 4 1.22E+03 2.68E+04 8.11E+04 3.92E+04 1.41E+03 3.57E+04 4.43E+04 1.14E+04
Colony 2,Replicate 1 4.16E+01 2.18E+04 4.58E+04 4.31E+04 1.15E+03 3.22E+04 3.74E+04 1.00E+04
Colony 2,Replicate 2 6.32E+02 2.38E+04 4.89E+04 4.42E+04 2.04E+03 3.38E+04 4.03E+04 1.21E+04
Colony 2,Replicate 3 1.16E+03 2.39E+04 4.85E+04 4.69E+04 2.16E+03 3.76E+04 3.90E+04 1.27E+04
Colony 2,Replicate 4 1.68E+03 2.37E+04 4.97E+04 4.71E+04 1.98E+03 3.63E+04 4.02E+04 1.17E+04

Fig.13 Fluorescence per Particle

Finally, we counted the colonies on each plate with fewer than 300 colonies. Then, we multiplied by the Final Dilution Factor: 8 . We recorded the CFU per 1mL of each OD600 = 0.1 culture.