Difference between revisions of "Team:Nanjing-China/InterLab"

Line 81: Line 81:
 
     <div class="sub">
 
     <div class="sub">
 
       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/InterLab">InterLab</a></ul></li></div>
 
       <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/InterLab">InterLab</a></ul></li></div>
 +
      <ul>
 +
    <li><a href="#ca">Calibration</a></li>
 +
            <li><a href="#c_m">Cell measurement</a></li></ul>
 
</div>
 
</div>
 
</div>
 
</div>
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         <ul>
 
         <ul>
 
         <li><a href="https://2018.igem.org/Team:Nanjing-China/Basic_Part">Basic_Part</a></li>
 
         <li><a href="https://2018.igem.org/Team:Nanjing-China/Basic_Part">Basic_Part</a></li>
−
            <li><a href="https://2018.igem.org/Team:Nanjing-China/Improved_Parts">Improved</a></li>
 
 
</ul>
 
</ul>
 
             </li>
 
             </li>
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                 <li><a href="https://2018.igem.org/Team:Nanjing-China/Results">Results</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Nanjing-China/Results">Results</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Nanjing-China/Demonstrate">Demonstrate</a></li>
 
                 <li><a href="https://2018.igem.org/Team:Nanjing-China/Demonstrate">Demonstrate</a></li>
−
             <li><a href="https://2018.igem.org/Team:Nanjing-China/Improvement">Improvement</a></li>
+
             <li><a href="https://2018.igem.org/Team:Nanjing-China/Device">Device</a></li>
 
</ul>
 
</ul>
 
         </li>
 
         </li>
Line 139: Line 141:
 
     <div class="word">
 
     <div class="word">
 
     <h1>InterLab</h1>
 
     <h1>InterLab</h1>
−
  <div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/d/dd/T--Nanjing-China--InterLab-1.jpg" width="90%" /></div>
+
    <p>Our  team Nanjing-China used <em>E. coli</em> K-12 DH5-alpha to conduct the InterLab Study. The instrument used during our measurements is  Tecan Infinite M1000 Pro plate reader which could read both fluorescence and absorbance  from the top of the plate. It has variable temperature settings and pathlength correction, which can be disabled.</p>
−
  <div class="word-1" style="height:30px;"></div>
+
    </div>
−
   <div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/c/c0/T--Nanjing-China--InterLab-2.jpg" width="90%" /></div>   
+
    <div class="word" id="ca">
−
     <div class="word-1" style="height:30px;"></div>
+
    <h2><u>Calibration  Protocols</u></h2>
−
<div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/4/4e/T--Nanjing-China--InterLab-3.jpg"width="90%"  /></div>
+
    <p>We used black plates with transparent bottom for the calibration measurements, which had flat-bottomed wells.</p>
−
    <div class="word-1" style="height:30px;"></div>
+
    <h3><u>Calibration 1: OD600 Reference point - LUDOX  Protocol</u></h3>
−
<div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/d/d9/T--Nanjing-China--InterLab-4.jpg"width="90%" /></div>
+
    <div class="word-1">
 +
    <table border="1" cellspacing="0" cellpadding="0">
 +
      <tr>
 +
        <td width="109" valign="bottom"><p>&nbsp;</p></td>
 +
        <td width="90" valign="bottom"><p align="center">LUDOX CL-X </p></td>
 +
        <td width="90" valign="bottom"><p align="center">H2O </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Replicate 1 </p></td>
 +
        <td width="90"><p align="center">0.0506 </p></td>
 +
        <td width="90"><p align="center">0.0372 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Replicate 2 </p></td>
 +
        <td width="90"><p align="center">0.050799999 </p></td>
 +
        <td width="90"><p align="center">0.0353 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Replicate 3 </p></td>
 +
        <td width="90"><p align="center">0.0517 </p></td>
 +
        <td width="90"><p align="center">0.033100002 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Replicate 4 </p></td>
 +
        <td width="90"><p align="center">0.050500002 </p></td>
 +
        <td width="90"><p align="center">0.034600001 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Arith. Mean </p></td>
 +
        <td width="90" valign="bottom"><p align="center">0.051 </p></td>
 +
        <td width="90" valign="bottom"><p align="center">0.035 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Corrected Abs600 </p></td>
 +
        <td width="90" valign="bottom"><p align="center">0.016 </p></td>
 +
        <td width="90"><p>&nbsp;</p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">Reference OD600 </p></td>
 +
        <td width="90" valign="bottom"><p align="center">0.074 </p></td>
 +
        <td width="90"><p>&nbsp;</p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="109" valign="bottom"><p align="left">OD600/Abs600 </p></td>
 +
        <td width="90" valign="bottom"><p align="center">4.669 </p></td>
 +
        <td width="90"><p>&nbsp;</p></td>
 +
      </tr>
 +
    </table>
 +
    <p><font size="-1">Figure 1. OD600 Reference point</font></p>
 +
    </div>
 +
    <p>We used LUDOX CL-X as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement. </p>
 +
    <h3><u>Calibration 2: Particle Standard Curve - Microsphere Protocol</u></h3>
 +
<div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/d/dd/T--Nanjing-China--InterLab-1.jpg" width="90%" />
 +
    <p><font size="-1">Figure 2. Particle Standard Curve</font></p>
 +
    </div>
 +
   <div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/c/c0/T--Nanjing-China--InterLab-2.jpg" width="90%" />
 +
      <p><font size="-1">Figure 3. Particle Standard Curve (log scale)</font></p>
 +
</div>   
 +
<p>We prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. The size and optical characteristics of these microspheres were similar to cells, and there was a known amount of particles per volume. This measurement would allow us to construct a standard curve of particle concentration which could be used to convert Abs<sub>600</sub> measurements to an estimated number of cells.</p>
 +
     <h3><u>Calibration 3: Fluorescence standard curve - Fluorescein Protocol</u></h3>
 +
<div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/4/4e/T--Nanjing-China--InterLab-3.jpg"width="90%"  />
 +
    <p><font size="-1">Figure 4. Fluorescein Standard Curve</font></p>
 +
    </div>
 +
<div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/d/d9/T--Nanjing-China--InterLab-4.jpg"width="90%"  />
 +
    <p><font size="-1">Figure 5. Fluorescein Standard Curve (log scale)</font></p>
 +
    </div>
 +
    <p>We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We would be able to use this to convert our cell based readings to an equivalent fluorescein concentration.</p>
 +
    </div>
 +
    <div class="word" id="c_m">
 +
    <h1><u>Cell measurement protocol</u></h1>
 +
    <p>After completing all three of the calibration measurements, we started performing the cell measurements. We use E. coli K-12 DH5-alpha strain, the same plates and volumes that we used in our calibration protocol, as well as the same settings (e.g., filters or excitation and emission wavelengths) that we used in our calibration measurements for the sake of consistence.</p>
 +
    <div class="word-1">
 +
    <table border="0" cellspacing="0" cellpadding="0" width="0">
 +
      <tr>
 +
        <td width="703" colspan="10" valign="bottom"><p><strong>Fluorescence Raw Readings:</strong></p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left"><strong>Hour 0:</strong><strong> </strong></p></td>
 +
        <td width="61" valign="bottom"><p align="left">Neg. Control </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Pos. Control </p></td>
 +
        <td width="56" valign="bottom"><p align="left">Device 1 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 2 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 3 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 4 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 5 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 6 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">LB + Chlor (blank) </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 1 </p></td>
 +
        <td width="61"><p align="left">502 </p></td>
 +
        <td width="62"><p align="left">1077 </p></td>
 +
        <td width="56"><p align="left">507 </p></td>
 +
        <td width="62"><p align="left">1112 </p></td>
 +
        <td width="62"><p align="left">490 </p></td>
 +
        <td width="62"><p align="left">1514 </p></td>
 +
        <td width="62"><p align="left">759 </p></td>
 +
        <td width="62"><p align="left">623 </p></td>
 +
        <td width="62"><p align="left">477 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 2 </p></td>
 +
        <td width="61"><p align="left">517 </p></td>
 +
        <td width="62"><p align="left">1177 </p></td>
 +
        <td width="56"><p align="left">530 </p></td>
 +
        <td width="62"><p align="left">1184 </p></td>
 +
        <td width="62"><p align="left">524 </p></td>
 +
        <td width="62"><p align="left">1683 </p></td>
 +
        <td width="62"><p align="left">842 </p></td>
 +
        <td width="62"><p align="left">656 </p></td>
 +
        <td width="62"><p align="left">500 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 3 </p></td>
 +
        <td width="61"><p align="left">492 </p></td>
 +
        <td width="62"><p align="left">1129 </p></td>
 +
        <td width="56"><p align="left">506 </p></td>
 +
        <td width="62"><p align="left">1118 </p></td>
 +
        <td width="62"><p align="left">489 </p></td>
 +
        <td width="62"><p align="left">1520 </p></td>
 +
        <td width="62"><p align="left">760 </p></td>
 +
        <td width="62"><p align="left">632 </p></td>
 +
        <td width="62"><p align="left">486 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 4 </p></td>
 +
        <td width="61"><p align="left">506 </p></td>
 +
        <td width="62"><p align="left">1202 </p></td>
 +
        <td width="56"><p align="left">556 </p></td>
 +
        <td width="62"><p align="left">1234 </p></td>
 +
        <td width="62"><p align="left">517 </p></td>
 +
        <td width="62"><p align="left">1709 </p></td>
 +
        <td width="62"><p align="left">760 </p></td>
 +
        <td width="62"><p align="left">664 </p></td>
 +
        <td width="62"><p align="left">499 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 1 </p></td>
 +
        <td width="61"><p align="left">488 </p></td>
 +
        <td width="62"><p align="left">1102 </p></td>
 +
        <td width="56"><p align="left">621 </p></td>
 +
        <td width="62"><p align="left">1368 </p></td>
 +
        <td width="62"><p align="left">508 </p></td>
 +
        <td width="62"><p align="left">1307 </p></td>
 +
        <td width="62"><p align="left">821 </p></td>
 +
        <td width="62"><p align="left">662 </p></td>
 +
        <td width="62"><p align="left">498 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 2 </p></td>
 +
        <td width="61"><p align="left">513 </p></td>
 +
        <td width="62"><p align="left">1165 </p></td>
 +
        <td width="56"><p align="left">635 </p></td>
 +
        <td width="62"><p align="left">1443 </p></td>
 +
        <td width="62"><p align="left">534 </p></td>
 +
        <td width="62"><p align="left">1444 </p></td>
 +
        <td width="62"><p align="left">979 </p></td>
 +
        <td width="62"><p align="left">706 </p></td>
 +
        <td width="62"><p align="left">483 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 3 </p></td>
 +
        <td width="61"><p align="left">490 </p></td>
 +
        <td width="62"><p align="left">1077 </p></td>
 +
        <td width="56"><p align="left">601 </p></td>
 +
        <td width="62"><p align="left">1360 </p></td>
 +
        <td width="62"><p align="left">508 </p></td>
 +
        <td width="62"><p align="left">1361 </p></td>
 +
        <td width="62"><p align="left">939 </p></td>
 +
        <td width="62"><p align="left">683 </p></td>
 +
        <td width="62"><p align="left">495 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 4 </p></td>
 +
        <td width="61"><p align="left">510 </p></td>
 +
        <td width="62"><p align="left">1135 </p></td>
 +
        <td width="56"><p align="left">621 </p></td>
 +
        <td width="62"><p align="left">1493 </p></td>
 +
        <td width="62"><p align="left">535 </p></td>
 +
        <td width="62"><p align="left">1380 </p></td>
 +
        <td width="62"><p align="left">972 </p></td>
 +
        <td width="62"><p align="left">601 </p></td>
 +
        <td width="62"><p align="left">480 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left"><strong>Hour 6:</strong><strong> </strong></p></td>
 +
        <td width="61" valign="bottom"><p align="left">Neg. Control </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Pos. Control </p></td>
 +
        <td width="56" valign="bottom"><p align="left">Device 1 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 2 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 3 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 4 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 5 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">Device 6 </p></td>
 +
        <td width="62" valign="bottom"><p align="left">LB + Chlor (blank) </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 1 </p></td>
 +
        <td width="61"><p align="left">564 </p></td>
 +
        <td width="62"><p align="left">2645 </p></td>
 +
        <td width="56"><p align="left">2277 </p></td>
 +
        <td width="62"><p align="left">2692 </p></td>
 +
        <td width="62"><p align="left">517 </p></td>
 +
        <td width="62"><p align="left">6432 </p></td>
 +
        <td width="62"><p align="left">4002 </p></td>
 +
        <td width="62"><p align="left">1131 </p></td>
 +
        <td width="62"><p align="left">465 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 2 </p></td>
 +
        <td width="61"><p align="left">569 </p></td>
 +
        <td width="62"><p align="left">2742 </p></td>
 +
        <td width="56"><p align="left">2536 </p></td>
 +
        <td width="62"><p align="left">2759 </p></td>
 +
        <td width="62"><p align="left">585 </p></td>
 +
        <td width="62"><p align="left">6076 </p></td>
 +
        <td width="62"><p align="left">4277 </p></td>
 +
        <td width="62"><p align="left">1114 </p></td>
 +
        <td width="62"><p align="left">452 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 3 </p></td>
 +
        <td width="61"><p align="left">592 </p></td>
 +
        <td width="62"><p align="left">2672 </p></td>
 +
        <td width="56"><p align="left">2449 </p></td>
 +
        <td width="62"><p align="left">2844 </p></td>
 +
        <td width="62"><p align="left">565 </p></td>
 +
        <td width="62"><p align="left">5808 </p></td>
 +
        <td width="62"><p align="left">4207 </p></td>
 +
        <td width="62"><p align="left">1094 </p></td>
 +
        <td width="62"><p align="left">453 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 1, Replicate 4 </p></td>
 +
        <td width="61"><p align="left">582 </p></td>
 +
        <td width="62"><p align="left">2718 </p></td>
 +
        <td width="56"><p align="left">2577 </p></td>
 +
        <td width="62"><p align="left">2927 </p></td>
 +
        <td width="62"><p align="left">590 </p></td>
 +
        <td width="62"><p align="left">6399 </p></td>
 +
        <td width="62"><p align="left">4286 </p></td>
 +
        <td width="62"><p align="left">1126 </p></td>
 +
        <td width="62"><p align="left">468 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 1 </p></td>
 +
        <td width="61"><p align="left">506 </p></td>
 +
        <td width="62"><p align="left">2577 </p></td>
 +
        <td width="56"><p align="left">3731 </p></td>
 +
        <td width="62"><p align="left">2690 </p></td>
 +
        <td width="62"><p align="left">548 </p></td>
 +
        <td width="62"><p align="left">7511 </p></td>
 +
        <td width="62"><p align="left">4772 </p></td>
 +
        <td width="62"><p align="left">1171 </p></td>
 +
        <td width="62"><p align="left">466 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 2 </p></td>
 +
        <td width="61"><p align="left">570 </p></td>
 +
        <td width="62"><p align="left">2772 </p></td>
 +
        <td width="56"><p align="left">4227 </p></td>
 +
        <td width="62"><p align="left">2903 </p></td>
 +
        <td width="62"><p align="left">603 </p></td>
 +
        <td width="62"><p align="left">7621 </p></td>
 +
        <td width="62"><p align="left">5307 </p></td>
 +
        <td width="62"><p align="left">1230 </p></td>
 +
        <td width="62"><p align="left">475 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 3 </p></td>
 +
        <td width="61"><p align="left">558 </p></td>
 +
        <td width="62"><p align="left">2662 </p></td>
 +
        <td width="56"><p align="left">3997 </p></td>
 +
        <td width="62"><p align="left">2762 </p></td>
 +
        <td width="62"><p align="left">575 </p></td>
 +
        <td width="62"><p align="left">7373 </p></td>
 +
        <td width="62"><p align="left">5061 </p></td>
 +
        <td width="62"><p align="left">1243 </p></td>
 +
        <td width="62"><p align="left">486 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">Colony 2, Replicate 4 </p></td>
 +
        <td width="61"><p align="left">572 </p></td>
 +
        <td width="62"><p align="left">2756 </p></td>
 +
        <td width="56"><p align="left">4160 </p></td>
 +
        <td width="62"><p align="left">2997 </p></td>
 +
        <td width="62"><p align="left">625 </p></td>
 +
        <td width="62"><p align="left">7555 </p></td>
 +
        <td width="62"><p align="left">5278 </p></td>
 +
        <td width="62"><p align="left">1299 </p></td>
 +
        <td width="62"><p align="left">473 </p></td>
 +
      </tr>
 +
      <tr>
 +
        <td width="149" valign="bottom"><p align="left">&nbsp;</p></td>
 +
        <td width="61"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
        <td width="56"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
        <td width="62"><p align="left">&nbsp;</p></td>
 +
      </tr>
 +
    </table>
 +
    <p><font size="-1">Figure 6. Fluorescence Raw Plate Readings</font></p>
 +
    </div>
 +
    <div class="word-1">
 +
      <table border="0" cellspacing="0" cellpadding="0" width="0">
 +
        <tr>
 +
          <td width="703" colspan="10" valign="bottom"><p><strong>Abs600 Raw Readings:</strong></p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left"><strong>Hour 0:</strong><strong> </strong></p></td>
 +
          <td width="63" valign="bottom"><p align="left">Neg. Control </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Pos. Control </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 1 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 2 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 3 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 4 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 5 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 6 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">LB + Chlor (blank) </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 1 </p></td>
 +
          <td width="63"><p align="left">0.0604 </p></td>
 +
          <td width="63"><p align="left">0.0583 </p></td>
 +
          <td width="63"><p align="left">0.0401 </p></td>
 +
          <td width="63"><p align="left">0.0594 </p></td>
 +
          <td width="63"><p align="left">0.0596 </p></td>
 +
          <td width="63"><p align="left">0.0565 </p></td>
 +
          <td width="63"><p align="left">0.0489 </p></td>
 +
          <td width="63"><p align="left">0.0613 </p></td>
 +
          <td width="63"><p align="left">0.0390 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 2 </p></td>
 +
          <td width="63"><p align="left">0.0604 </p></td>
 +
          <td width="63"><p align="left">0.0654 </p></td>
 +
          <td width="63"><p align="left">0.0395 </p></td>
 +
          <td width="63"><p align="left">0.0601 </p></td>
 +
          <td width="63"><p align="left">0.0598 </p></td>
 +
          <td width="63"><p align="left">0.0583 </p></td>
 +
          <td width="63"><p align="left">0.0499 </p></td>
 +
          <td width="63"><p align="left">0.0603 </p></td>
 +
          <td width="63"><p align="left">0.0380 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 3 </p></td>
 +
          <td width="63"><p align="left">0.0634 </p></td>
 +
          <td width="63"><p align="left">0.0602 </p></td>
 +
          <td width="63"><p align="left">0.0429 </p></td>
 +
          <td width="63"><p align="left">0.0633 </p></td>
 +
          <td width="63"><p align="left">0.0615 </p></td>
 +
          <td width="63"><p align="left">0.0586 </p></td>
 +
          <td width="63"><p align="left">0.0528 </p></td>
 +
          <td width="63"><p align="left">0.0649 </p></td>
 +
          <td width="63"><p align="left">0.0416 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 4 </p></td>
 +
          <td width="63"><p align="left">0.0527 </p></td>
 +
          <td width="63"><p align="left">0.0541 </p></td>
 +
          <td width="63"><p align="left">0.0365 </p></td>
 +
          <td width="63"><p align="left">0.0560 </p></td>
 +
          <td width="63"><p align="left">0.0570 </p></td>
 +
          <td width="63"><p align="left">0.0538 </p></td>
 +
          <td width="63"><p align="left">0.0436 </p></td>
 +
          <td width="63"><p align="left">0.0550 </p></td>
 +
          <td width="63"><p align="left">0.0338 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 1 </p></td>
 +
          <td width="63"><p align="left">0.0667 </p></td>
 +
          <td width="63"><p align="left">0.0670 </p></td>
 +
          <td width="63"><p align="left">0.0440 </p></td>
 +
          <td width="63"><p align="left">0.0763 </p></td>
 +
          <td width="63"><p align="left">0.0654 </p></td>
 +
          <td width="63"><p align="left">0.0535 </p></td>
 +
          <td width="63"><p align="left">0.0535 </p></td>
 +
          <td width="63"><p align="left">0.0621 </p></td>
 +
          <td width="63"><p align="left">0.0391 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 2 </p></td>
 +
          <td width="63"><p align="left">0.0704 </p></td>
 +
          <td width="63"><p align="left">0.0687 </p></td>
 +
          <td width="63"><p align="left">0.0420 </p></td>
 +
          <td width="63"><p align="left">0.0743 </p></td>
 +
          <td width="63"><p align="left">0.0647 </p></td>
 +
          <td width="63"><p align="left">0.0551 </p></td>
 +
          <td width="63"><p align="left">0.0545 </p></td>
 +
          <td width="63"><p align="left">0.0622 </p></td>
 +
          <td width="63"><p align="left">0.0391 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 3 </p></td>
 +
          <td width="63"><p align="left">0.0643 </p></td>
 +
          <td width="63"><p align="left">0.0651 </p></td>
 +
          <td width="63"><p align="left">0.0413 </p></td>
 +
          <td width="63"><p align="left">0.0698 </p></td>
 +
          <td width="63"><p align="left">0.0623 </p></td>
 +
          <td width="63"><p align="left">0.0591 </p></td>
 +
          <td width="63"><p align="left">0.0532 </p></td>
 +
          <td width="63"><p align="left">0.0610 </p></td>
 +
          <td width="63"><p align="left">0.0387 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 4 </p></td>
 +
          <td width="63"><p align="left">0.0601 </p></td>
 +
          <td width="63"><p align="left">0.0619 </p></td>
 +
          <td width="63"><p align="left">0.0369 </p></td>
 +
          <td width="63"><p align="left">0.0658 </p></td>
 +
          <td width="63"><p align="left">0.0596 </p></td>
 +
          <td width="63"><p align="left">0.0495 </p></td>
 +
          <td width="63"><p align="left">0.0477 </p></td>
 +
          <td width="63"><p align="left">0.0652 </p></td>
 +
          <td width="63"><p align="left">0.0448 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left"><strong>Hour 6:</strong><strong> </strong></p></td>
 +
          <td width="63" valign="bottom"><p align="left">Neg. Control </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Pos. Control </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 1 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 2 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 3 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 4 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 5 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">Device 6 </p></td>
 +
          <td width="63" valign="bottom"><p align="left">LB + Chlor (blank) </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 1 </p></td>
 +
          <td width="63"><p align="left">0.2471 </p></td>
 +
          <td width="63"><p align="left">0.2457 </p></td>
 +
          <td width="63"><p align="left">0.1465 </p></td>
 +
          <td width="63"><p align="left">0.2426 </p></td>
 +
          <td width="63"><p align="left">0.2047 </p></td>
 +
          <td width="63"><p align="left">0.0992 </p></td>
 +
          <td width="63"><p align="left">0.2124 </p></td>
 +
          <td width="63"><p align="left">0.2152 </p></td>
 +
          <td width="63"><p align="left">0.0384 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 2 </p></td>
 +
          <td width="63"><p align="left">0.2451 </p></td>
 +
          <td width="63"><p align="left">0.2410 </p></td>
 +
          <td width="63"><p align="left">0.1536 </p></td>
 +
          <td width="63"><p align="left">0.2435 </p></td>
 +
          <td width="63"><p align="left">0.2155 </p></td>
 +
          <td width="63"><p align="left">0.0936 </p></td>
 +
          <td width="63"><p align="left">0.2201 </p></td>
 +
          <td width="63"><p align="left">0.2046 </p></td>
 +
          <td width="63"><p align="left">0.0380 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 3 </p></td>
 +
          <td width="63"><p align="left">0.2467 </p></td>
 +
          <td width="63"><p align="left">0.2410 </p></td>
 +
          <td width="63"><p align="left">0.1541 </p></td>
 +
          <td width="63"><p align="left">0.2535 </p></td>
 +
          <td width="63"><p align="left">0.2139 </p></td>
 +
          <td width="63"><p align="left">0.0920 </p></td>
 +
          <td width="63"><p align="left">0.2125 </p></td>
 +
          <td width="63"><p align="left">0.2025 </p></td>
 +
          <td width="63"><p align="left">0.0412 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 1, Replicate 4 </p></td>
 +
          <td width="63"><p align="left">0.2391 </p></td>
 +
          <td width="63"><p align="left">0.2315 </p></td>
 +
          <td width="63"><p align="left">0.1500 </p></td>
 +
          <td width="63"><p align="left">0.2437 </p></td>
 +
          <td width="63"><p align="left">0.2051 </p></td>
 +
          <td width="63"><p align="left">0.0885 </p></td>
 +
          <td width="63"><p align="left">0.2081 </p></td>
 +
          <td width="63"><p align="left">0.1980 </p></td>
 +
          <td width="63"><p align="left">0.0327 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 1 </p></td>
 +
          <td width="63"><p align="left">0.2345 </p></td>
 +
          <td width="63"><p align="left">0.2556 </p></td>
 +
          <td width="63"><p align="left">0.2119 </p></td>
 +
          <td width="63"><p align="left">0.2481 </p></td>
 +
          <td width="63"><p align="left">0.2537 </p></td>
 +
          <td width="63"><p align="left">0.2062 </p></td>
 +
          <td width="63"><p align="left">0.2615 </p></td>
 +
          <td width="63"><p align="left">0.2253 </p></td>
 +
          <td width="63"><p align="left">0.0389 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 2 </p></td>
 +
          <td width="63"><p align="left">0.2604 </p></td>
 +
          <td width="63"><p align="left">0.2684 </p></td>
 +
          <td width="63"><p align="left">0.2357 </p></td>
 +
          <td width="63"><p align="left">0.2631 </p></td>
 +
          <td width="63"><p align="left">0.2746 </p></td>
 +
          <td width="63"><p align="left">0.1958 </p></td>
 +
          <td width="63"><p align="left">0.2798 </p></td>
 +
          <td width="63"><p align="left">0.2302 </p></td>
 +
          <td width="63"><p align="left">0.0386 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 3 </p></td>
 +
          <td width="63"><p align="left">0.2532 </p></td>
 +
          <td width="63"><p align="left">0.2640 </p></td>
 +
          <td width="63"><p align="left">0.2202 </p></td>
 +
          <td width="63"><p align="left">0.2516 </p></td>
 +
          <td width="63"><p align="left">0.2643 </p></td>
 +
          <td width="63"><p align="left">0.1922 </p></td>
 +
          <td width="63"><p align="left">0.2676 </p></td>
 +
          <td width="63"><p align="left">0.2323 </p></td>
 +
          <td width="63"><p align="left">0.0410 </p></td>
 +
        </tr>
 +
        <tr>
 +
          <td width="134" valign="bottom"><p align="left">Colony 2, Replicate 4 </p></td>
 +
          <td width="63"><p align="left">0.2525 </p></td>
 +
          <td width="63"><p align="left">0.2607 </p></td>
 +
          <td width="63"><p align="left">0.2241 </p></td>
 +
          <td width="63"><p align="left">0.2612 </p></td>
 +
          <td width="63"><p align="left">0.2682 </p></td>
 +
          <td width="63"><p align="left">0.1844 </p></td>
 +
          <td width="63"><p align="left">0.2632 </p></td>
 +
          <td width="63"><p align="left">0.2281 </p></td>
 +
          <td width="63"><p align="left">0.0336 </p></td>
 +
        </tr>
 +
      </table>
 +
      <p><font size="-1">Figure 7. Abs600 Raw Plate Readings</font></p>
 +
    </div>
 +
    <h3><u>Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures</u></h3>
 +
    <p>This procedure can be used to calibrate OD600  to colony forming unit (CFU) counts, which are directly related to the cell  concentration of the culture, i.e. viable cell counts per mL. This protocol  assumes that 1 bacterial cell will give rise to 1 colony.<br />
 +
      For the CFU protocol, we counted colonies for our two  Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040)  cultures.</p>
 +
      <div class="word-1" align="center"><img src="https://static.igem.org/mediawiki/2018/3/3d/T--Nanjing-China--result-1.png" width="70%" />
 +
        <p><font size="-1">Figure 8. Colony Forming Unit Calculations for Each Culture</font></p></div>
 +
      <p align="left">Based on the assumption that 1 bacterial cell gives  rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture  can be calculated as follows:</p>
 +
        <ol>
 +
          <li>Count the  colonies on each plate with fewer than 300 colonies.  </li>
 +
          <li>Multiple the colony count by  the Final Dilution Factor on each plate. </li>
 +
        </ol>
 
     </div>
 
     </div>
 
     </div>
 
     </div>
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         </div>
 
         </div>
 
       </div>
 
       </div>
−
      </div>
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Revision as of 11:07, 16 September 2018

Nanjing-China2018

InterLab

Our team Nanjing-China used E. coli K-12 DH5-alpha to conduct the InterLab Study. The instrument used during our measurements is Tecan Infinite M1000 Pro plate reader which could read both fluorescence and absorbance from the top of the plate. It has variable temperature settings and pathlength correction, which can be disabled.

Calibration Protocols

We used black plates with transparent bottom for the calibration measurements, which had flat-bottomed wells.

Calibration 1: OD600 Reference point - LUDOX Protocol

 

LUDOX CL-X

H2O

Replicate 1

0.0506

0.0372

Replicate 2

0.050799999

0.0353

Replicate 3

0.0517

0.033100002

Replicate 4

0.050500002

0.034600001

Arith. Mean

0.051

0.035

Corrected Abs600

0.016

 

Reference OD600

0.074

 

OD600/Abs600

4.669

 

Figure 1. OD600 Reference point

We used LUDOX CL-X as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement.

Calibration 2: Particle Standard Curve - Microsphere Protocol

Figure 2. Particle Standard Curve

Figure 3. Particle Standard Curve (log scale)

We prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. The size and optical characteristics of these microspheres were similar to cells, and there was a known amount of particles per volume. This measurement would allow us to construct a standard curve of particle concentration which could be used to convert Abs600 measurements to an estimated number of cells.

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Figure 4. Fluorescein Standard Curve

Figure 5. Fluorescein Standard Curve (log scale)

We prepared a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We would be able to use this to convert our cell based readings to an equivalent fluorescein concentration.

Cell measurement protocol

After completing all three of the calibration measurements, we started performing the cell measurements. We use E. coli K-12 DH5-alpha strain, the same plates and volumes that we used in our calibration protocol, as well as the same settings (e.g., filters or excitation and emission wavelengths) that we used in our calibration measurements for the sake of consistence.

Fluorescence Raw Readings:

Hour 0:

Neg. Control

Pos. Control

Device 1

Device 2

Device 3

Device 4

Device 5

Device 6

LB + Chlor (blank)

Colony 1, Replicate 1

502

1077

507

1112

490

1514

759

623

477

Colony 1, Replicate 2

517

1177

530

1184

524

1683

842

656

500

Colony 1, Replicate 3

492

1129

506

1118

489

1520

760

632

486

Colony 1, Replicate 4

506

1202

556

1234

517

1709

760

664

499

Colony 2, Replicate 1

488

1102

621

1368

508

1307

821

662

498

Colony 2, Replicate 2

513

1165

635

1443

534

1444

979

706

483

Colony 2, Replicate 3

490

1077

601

1360

508

1361

939

683

495

Colony 2, Replicate 4

510

1135

621

1493

535

1380

972

601

480

Hour 6:

Neg. Control

Pos. Control

Device 1

Device 2

Device 3

Device 4

Device 5

Device 6

LB + Chlor (blank)

Colony 1, Replicate 1

564

2645

2277

2692

517

6432

4002

1131

465

Colony 1, Replicate 2

569

2742

2536

2759

585

6076

4277

1114

452

Colony 1, Replicate 3

592

2672

2449

2844

565

5808

4207

1094

453

Colony 1, Replicate 4

582

2718

2577

2927

590

6399

4286

1126

468

Colony 2, Replicate 1

506

2577

3731

2690

548

7511

4772

1171

466

Colony 2, Replicate 2

570

2772

4227

2903

603

7621

5307

1230

475

Colony 2, Replicate 3

558

2662

3997

2762

575

7373

5061

1243

486

Colony 2, Replicate 4

572

2756

4160

2997

625

7555

5278

1299

473

 

 

 

 

 

 

 

 

 

 

Figure 6. Fluorescence Raw Plate Readings

Abs600 Raw Readings:

Hour 0:

Neg. Control

Pos. Control

Device 1

Device 2

Device 3

Device 4

Device 5

Device 6

LB + Chlor (blank)

Colony 1, Replicate 1

0.0604

0.0583

0.0401

0.0594

0.0596

0.0565

0.0489

0.0613

0.0390

Colony 1, Replicate 2

0.0604

0.0654

0.0395

0.0601

0.0598

0.0583

0.0499

0.0603

0.0380

Colony 1, Replicate 3

0.0634

0.0602

0.0429

0.0633

0.0615

0.0586

0.0528

0.0649

0.0416

Colony 1, Replicate 4

0.0527

0.0541

0.0365

0.0560

0.0570

0.0538

0.0436

0.0550

0.0338

Colony 2, Replicate 1

0.0667

0.0670

0.0440

0.0763

0.0654

0.0535

0.0535

0.0621

0.0391

Colony 2, Replicate 2

0.0704

0.0687

0.0420

0.0743

0.0647

0.0551

0.0545

0.0622

0.0391

Colony 2, Replicate 3

0.0643

0.0651

0.0413

0.0698

0.0623

0.0591

0.0532

0.0610

0.0387

Colony 2, Replicate 4

0.0601

0.0619

0.0369

0.0658

0.0596

0.0495

0.0477

0.0652

0.0448

Hour 6:

Neg. Control

Pos. Control

Device 1

Device 2

Device 3

Device 4

Device 5

Device 6

LB + Chlor (blank)

Colony 1, Replicate 1

0.2471

0.2457

0.1465

0.2426

0.2047

0.0992

0.2124

0.2152

0.0384

Colony 1, Replicate 2

0.2451

0.2410

0.1536

0.2435

0.2155

0.0936

0.2201

0.2046

0.0380

Colony 1, Replicate 3

0.2467

0.2410

0.1541

0.2535

0.2139

0.0920

0.2125

0.2025

0.0412

Colony 1, Replicate 4

0.2391

0.2315

0.1500

0.2437

0.2051

0.0885

0.2081

0.1980

0.0327

Colony 2, Replicate 1

0.2345

0.2556

0.2119

0.2481

0.2537

0.2062

0.2615

0.2253

0.0389

Colony 2, Replicate 2

0.2604

0.2684

0.2357

0.2631

0.2746

0.1958

0.2798

0.2302

0.0386

Colony 2, Replicate 3

0.2532

0.2640

0.2202

0.2516

0.2643

0.1922

0.2676

0.2323

0.0410

Colony 2, Replicate 4

0.2525

0.2607

0.2241

0.2612

0.2682

0.1844

0.2632

0.2281

0.0336

Figure 7. Abs600 Raw Plate Readings

Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

This procedure can be used to calibrate OD600 to colony forming unit (CFU) counts, which are directly related to the cell concentration of the culture, i.e. viable cell counts per mL. This protocol assumes that 1 bacterial cell will give rise to 1 colony.
For the CFU protocol, we counted colonies for our two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.

Figure 8. Colony Forming Unit Calculations for Each Culture

Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:

  1. Count the colonies on each plate with fewer than 300 colonies. 
  2. Multiple the colony count by the Final Dilution Factor on each plate.

Address

Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046

Email

nanjing_china@163.com

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