Difference between revisions of "Team:ECUST/InterLab"

 
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<center><specialh1 style="font-size: 75px; text-transform: lowercase;">
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            interlab
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<div class="container" style="width: 80% ; ">
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<img src="https://static.igem.org/mediawiki/2017/3/3d/Interlab.png" width="100%"/>
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    <h1>Background</h1>
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    <p>All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.</p>
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      <br><h1 hidden>Background</h1>
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          <h1 id="tables">Background</h1>
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<p>All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.</p>
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    <p>Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be continuously monitored without disrupting cells.</p>
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    <p>First, we measured the standard curve by diluting supplied FITC. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.</p>
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    <p>After making the standard curve, we successfully transformed eight plasmids provided by Measurement Committee and do the cell measurement according to the protocol.</p>
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          <h1 id="tables">Overview</h1>
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<div class="style1">
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    <p>Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be continuously monitored without disrupting cells.</p>
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    <p>First, we measured the standard curve by diluting supplied FITC. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.</p>
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    <p>After making the standard curve, we successfully transformed eight plasmids provided by Measurement Committee and do the cell measurement according to the protocol.</p>
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  <h1>Materials and methods</h1><br><br>
 
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  <div >
 
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  <p><b>plasmid used</b></p><br>
 
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  <p style="margin-left: 20px;">
 
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Positive control<br>
 
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Negative control<br>
 
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Test Device 1: J23101.BCD2.E0040.B0015<br>
 
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Test Device 2: J23106.BCD2.E0040.B0015<br>
 
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Test Device 3: J23117.BCD2.E0040.B0015<br>
 
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Test Device 4: J23101+I13054<br>
 
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Test Device 5: J23106+I13054<br>
 
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Test Device 6: J23117+I13054<br></p>
 
  
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<p><b>Strain used</b></p>
 
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<p style="margin-left: 20px;"><i>Escherichia coli</i> DH5a</p>
 
  
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<p><b>Materials</b></p>
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      <br><h1 hidden>Materials and methods</h1>
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LUDOX<br>
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H20<br>
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black flat bottom 96 well plate<br>
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fluorescein<br>
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1xPBS<br>
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LB(Luria Bertani) media<br>
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Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)<br>
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50ml Falcon tube<br></p>
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<p><b>Machines</b></p>
 
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<p style="margin-left: 20px;">
 
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Clariostar -430 -9903<br>
 
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thermostatic shaker THZ.C<br>
 
−
electro-heating standing-temperature cultivator DHP-9082<br></p>
 
  
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          <h1 id="tables">Materials and methods</h1>
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  <div >
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  <p><b>plasmid used</b></p><br>
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  <p style="margin-left: 20px;">
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Positive control<br>
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Negative control<br>
 +
Test Device 1: J23101.BCD2.E0040.B0015<br>
 +
Test Device 2: J23106.BCD2.E0040.B0015<br>
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Test Device 3: J23117.BCD2.E0040.B0015<br>
 +
Test Device 4: J23101+I13054<br>
 +
Test Device 5: J23106+I13054<br>
 +
Test Device 6: J23117+I13054<br></p>
  
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<p><b>Methods</b></p>
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<p><b>Strain used</b></p>
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<p style="margin-left: 20px;">
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<p style="margin-left: 20px;"><i>Escherichia coli</i> DH5a</p>
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<a href="">InterLab 2017 Plate Reader Protocol</a>
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</p>
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<p><b>Materials</b></p>
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  </div>
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<p style="margin-left: 20px;">
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LUDOX<br>
 +
H20<br>
 +
black flat bottom 96 well plate<br>
 +
fluorescein<br>
 +
1xPBS<br>
 +
LB(Luria Bertani) media<br>
 +
Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)<br>
 +
50ml Falcon tube<br></p>
  
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<div class="some-padding"></div>
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<p><b>Machines</b></p>
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<div class="some-padding"></div>
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<p style="margin-left: 20px;">
 +
Clariostar -430 -9903<br>
 +
thermostatic shaker THZ.C<br>
 +
electro-heating standing-temperature cultivator DHP-9082<br></p>
  
  
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<div class="row">
+
<p><b>Methods</b></p>
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<h1>Results</h1>
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<p style="margin-left: 20px;">
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<div class="some-padding"></div>
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<a href="">InterLab 2017 Plate Reader Protocol</a>
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<h3>1. Stand curve</h3><br><br>
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</p>
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  <div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/7/7b/Int1.png" alt="" style="width: 600px;"> </div>
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  <div style="font-size:10px;">
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  <p>Table 1. Data of FITC standard curve</p></div>
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  <img src="https://static.igem.org/mediawiki/2017/c/cc/Int1a.png" alt="">
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  <center>
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  <div style="font-size:10px;">
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  <p>Figure 1a. FITC standard curve（absolute）</p></div><br><br><br><br>
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  <img src="https://static.igem.org/mediawiki/2017/a/a6/Int1b.png" alt="">
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  <center>
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  <div style="font-size:10px;">
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  <p> Figure 1b. FITC standard curve（log）</p></div><br><br><br><br>
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<div class="some-padding"></div>
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<h3>2. Transformation</h3>
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  <img src="https://static.igem.org/mediawiki/2017/e/ec/Int2.png" alt="">
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  <center>
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  <div style="font-size:10px;">
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  <p>Figure 2. Verification by Colony PCR</p></div><br><br><br><br>
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  </center>
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  <div class="col-md-6">
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  <p>
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P1,P2：two clones of positive control<br>
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−
N1,N2：two clones of Negative control<br>
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1-1,1-2：two clones of Device1<br>
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2-1,2-2：two clones of Device2<br>
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3-1,3-2：two clones of Device3<br>
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−
4-1,4-2：two clones of Device4<br>
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5-1,5-2：two clones of Device5<br>
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6-1,6-2：two clones of Device6<br></p>
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<div class="some-padding"></div>
 
  
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<h3>3. Cell&nbsp;Measurement</h3><br><br>
 
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<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/8/8a/Intt2.png" alt="" style="width: 600px;"> </div>
 
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  <center>
 
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  <div style="font-size:10px;">
 
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  <p>Table 2.  Data of OD600（clone 1）.</p></div><br><br><br>
 
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<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/d/dd/Intt3.png" alt="" style="width: 600px;"> </div>
 
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  <center>
 
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  <div style="font-size:10px;">
 
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  <p>Table 3. Data of OD600（clone 2）</p></div><br><br><br>
 
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      <br><h1 hidden>Results</h1>
  
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<div class="col-md-6" align="center">
 
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  <img src="https://static.igem.org/mediawiki/2017/2/2d/Int3.png" alt="">
 
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  <center>
 
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  <div style="font-size:10px;">
 
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  <p>Figure 3.curve of OD600（clone 1）</p></div><br><br>
 
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  <img src="https://static.igem.org/mediawiki/2017/8/8f/Int4.png" alt="">
 
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  <div style="font-size:10px;">
 
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  <p>Figure 4.curve of OD600（clone 2）</p></div><br><br>
 
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<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/2/2d/Intt4.png" alt="" style="width: 600px;"> </div>
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          <h1 id="tables">Results</h1>
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  <p>Table 4. Data of Fluorescence（clone 1）</p></div><br><br>
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        </div>
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  </center>
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<div class="row">
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<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/c/cc/Intt5.png" alt="" style="width: 600px;"> </div>
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  <center>
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<div class="some-padding"></div>
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  <div style="font-size:10px;">
+
<h3>1. Stand curve</h3><br><br>
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  <p>Table 5. Data of Fluorescence（clone 2）</p></div><br><br>
+
  <div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/7/7b/Int1.png" alt="" style="width: 600px;"> </div>
−
  </center>
+
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Table 1. Data of FITC standard curve</p></div>
 +
  </center>
 +
  <div class="some-padding"></div>
 +
 
 +
  <div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/c/cc/Int1a.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Figure 1a. FITC standard curve（absolute）</p></div><br><br><br><br>
 +
  </center>
 +
  </div>
 +
  <div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/a/a6/Int1b.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p> Figure 1b. FITC standard curve（log）</p></div><br><br><br><br>
 +
  </center>
 +
  </div>
 +
<div class="some-padding"></div>
 +
<div class="some-padding"></div>
 +
 
 +
 
 +
<h3>2. Transformation</h3>
 +
  <div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/e/ec/Int2.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Figure 2. Verification by Colony PCR</p></div><br><br><br><br>
 +
  </center>
 +
  </div>
 +
  <div class="col-md-6">
 +
  <p>
 +
P1,P2：two clones of positive control<br>
 +
N1,N2：two clones of Negative control<br>
 +
1-1,1-2：two clones of Device1<br>
 +
2-1,2-2：two clones of Device2<br>
 +
3-1,3-2：two clones of Device3<br>
 +
4-1,4-2：two clones of Device4<br>
 +
5-1,5-2：two clones of Device5<br>
 +
6-1,6-2：two clones of Device6<br></p>
 +
 
 +
  </div>
 +
<br>
 +
<div class="some-padding"></div>
 +
 
 +
<h3>3. Cell&nbsp;Measurement</h3><br><br>
 +
<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/8/8a/Intt2.png" alt="" style="width: 600px;"> </div>
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Table 2.  Data of OD600（clone 1）.</p></div><br><br><br>
 +
  </center>
 +
<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/d/dd/Intt3.png" alt="" style="width: 600px;"> </div>
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Table 3. Data of OD600（clone 2）</p></div><br><br><br>
 +
  </center>
 +
 
 +
 
 +
<div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/2/2d/Int3.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Figure 3.curve of OD600（clone 1）</p></div><br><br>
 +
  </center>
 +
  </div>
 +
  <div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/8/8f/Int4.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Figure 4.curve of OD600（clone 2）</p></div><br><br>
 +
  </center>
 +
  </div>
 +
 +
 
 +
<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/2/2d/Intt4.png" alt="" style="width: 600px;"> </div>
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Table 4. Data of Fluorescence（clone 1）</p></div><br><br>
 +
  </center>
 +
<div class="row" align="center"> <img src="https://static.igem.org/mediawiki/2017/c/cc/Intt5.png" alt="" style="width: 600px;"> </div>
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Table 5. Data of Fluorescence（clone 2）</p></div><br><br>
 +
  </center>
 +
 
 +
 
 +
<div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/5/5e/Int5.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Figure 5.curve of Fluorescence（clone 1）</p></div><br><br>
 +
  </center>
 +
  </div>
 +
  <div class="col-md-6" align="center">
 +
  <img src="https://static.igem.org/mediawiki/2017/6/6d/Int6.png" alt="">
 +
  <center>
 +
  <div style="font-size:10px;">
 +
  <p>Figure 6.curve of Fluorescence（clone 2）</p></div><br><br>
 +
  </center>
 +
  </div>
 +
 
 +
</div>
 +
  </div>
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      <br><h1 hidden>Discussion</h1>
 +
 
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      <div class="row">
 +
        <div class="page-header">
 +
          <h1 id="tables">Discussion</h1>
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        </div>
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<div class="row">
 +
 
 +
<specialh5>Compare (Device 1, Device 2, Device 3) or (Device 4, Device 5, Device 6)</specialh5>
 +
<p>
 +
&nbsp;&nbsp;1. It is noticeable that the promoter of Device 1or4 is strongest followed by Device 2or5, Device3or6. <br>
 +
&nbsp;&nbsp;2. E.coli transformed by Device 1 grew slowly due to the high expression of GFP while those transformed by other Devices has similar growth rate. <br><br>
 +
</p>
 +
 +
 
 +
<specialh5>Compare (Device 1 and Device 4), (Device 2 and Device 5), (Device 3 and Device 6).</specialh5>
 +
<p >&nbsp;&nbsp;RBS of Device 1,2,3 is stronger than those of Device 4,5,6.</p>
 +
 
 +
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 +
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    <div class="some-padding"></div>
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<div class="col-md-6" align="center">
 
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  <img src="https://static.igem.org/mediawiki/2017/5/5e/Int5.png" alt="">
 
−
  <center>
 
−
  <div style="font-size:10px;">
 
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  <p>Figure 5.curve of Fluorescence（clone 1）</p></div><br><br>
 
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  <p>Figure 6.curve of Fluorescence（clone 2）</p></div><br><br>
 
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<h1>DISCUSSION</h1>
 
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<h5>Compare (Device 1, Device 2, Device 3) or (Device 4, Device 5, Device 6)</h5>
 
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<ol style="font-size:16px;">
 
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<li>It is noticeable that the promoter of Device 1or4 is strongest followed by Device 2or5, Device3or6.</li>
 
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<li>E.coli transformed by Device 1 grew slowly due to the high expression of GFP while those transformed by other Devices has similar growth rate.</li>
 
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</ol>
 
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<h5>Compare (Device 1 and Device 4), (Device 2 and Device 5), (Device 3 and Device 6).</h5>
 
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<p style="margin-left: 20px;">RBS of Device 1,2,3 is stronger than those of Device 4,5,6.</p>
 
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Latest revision as of 03:30, 2 November 2017



Background

Background

All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.


Overview

Overview

Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be continuously monitored without disrupting cells.

First, we measured the standard curve by diluting supplied FITC. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.

After making the standard curve, we successfully transformed eight plasmids provided by Measurement Committee and do the cell measurement according to the protocol.


Materials and methods

Materials and methods

plasmid used


Positive control
Negative control
Test Device 1: J23101.BCD2.E0040.B0015
Test Device 2: J23106.BCD2.E0040.B0015
Test Device 3: J23117.BCD2.E0040.B0015
Test Device 4: J23101+I13054
Test Device 5: J23106+I13054
Test Device 6: J23117+I13054

Strain used

Escherichia coli DH5a

Materials

LUDOX
H20
black flat bottom 96 well plate
fluorescein
1xPBS
LB(Luria Bertani) media
Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)
50ml Falcon tube

Machines

Clariostar -430 -9903
thermostatic shaker THZ.C
electro-heating standing-temperature cultivator DHP-9082

Methods

InterLab 2017 Plate Reader Protocol


Results

Results

1. Stand curve



Table 1. Data of FITC standard curve

Figure 1a. FITC standard curve（absolute）





Figure 1b. FITC standard curve（log）





2. Transformation

Figure 2. Verification by Colony PCR





P1,P2：two clones of positive control
N1,N2：two clones of Negative control
1-1,1-2：two clones of Device1
2-1,2-2：two clones of Device2
3-1,3-2：two clones of Device3
4-1,4-2：two clones of Device4
5-1,5-2：two clones of Device5
6-1,6-2：two clones of Device6


3. Cell Measurement



Table 2. Data of OD600（clone 1）.




Table 3. Data of OD600（clone 2）




Figure 3.curve of OD600（clone 1）



Figure 4.curve of OD600（clone 2）



Table 4. Data of Fluorescence（clone 1）



Table 5. Data of Fluorescence（clone 2）



Figure 5.curve of Fluorescence（clone 1）



Figure 6.curve of Fluorescence（clone 2）




Discussion

Discussion

Compare (Device 1, Device 2, Device 3) or (Device 4, Device 5, Device 6)

  1. It is noticeable that the promoter of Device 1or4 is strongest followed by Device 2or5, Device3or6.
  2. E.coli transformed by Device 1 grew slowly due to the high expression of GFP while those transformed by other Devices has similar growth rate.

Compare (Device 1 and Device 4), (Device 2 and Device 5), (Device 3 and Device 6).

  RBS of Device 1,2,3 is stronger than those of Device 4,5,6.