Difference between revisions of "Team:SDU CHINA/interlab"

 
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{{SDU_CHINA}}
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<br><br><br><br><br>
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<br><br>
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<div class="top"><img src="https://static.igem.org/mediawiki/2017/4/41/Interlabtop.jpeg"></div>
 +
 
 +
<div class="all">
 +
 
 
<div>
 
<div>
−
<h3>Background</h3>
+
<div class="title" >
−
<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 be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.</p>
+
  <strong style="color:#05BAC3"> Background</strong>
 +
  <p>&nbsp;</p>
 
</div>
 
</div>
−
 
+
<div class="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 be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.</div>
−
<br><br><br>
+
</div>
 +
<br><br><br><br>
  
 
<div>
 
<div>
−
<h3>Design</h3>
+
<div class="title" >
−
<p>We often used fluorescence as an indicator of promoter activity. However, the ability to repeat measurements in different labs has been difficult. To solve this problem, InterLab study has been developed as a robust measurement procedure for green fluorescent protein (GFP) which is one of the most used markers in synthetic biology.</p>
+
<strong style="color:#D5292C"> Design</strong>
−
<p>This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.</p>
+
  <p>&nbsp;</p>
 
</div>
 
</div>
−
 
+
<div class="p">We often used fluorescence as an indicator of promoter activity. However, the ability to repeat measurements in different labs has been difficult. To solve this problem, InterLab study has been developed as a robust measurement procedure for green fluorescent protein (GFP) which is one of the most used markers in synthetic biology.</div>
−
<br><br><br>
+
<div class="p">This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.</div>
 +
</div>
 +
<br><br><br><br>
  
 
<div>
 
<div>
−
<h3>Materials</h3>
+
<div class="title" ><strong style="color:#05BAC3">Materials</strong></div>
−
<h5>Plasmids used</h5>
+
<br><br>
−
<ul>
+
<div class="subtitle" style="color:#05BAC3" ><strong>Plasmids used</strong></div>
 +
<div class="p">
 +
<ul style="list-style:none">
 
<li>Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)</li>
 
<li>Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)</li>
 
<li>Negative Control Device: R0040 in pSB1C3 (Located in Kit Plate 6, well 20D)</li>
 
<li>Negative Control Device: R0040 in pSB1C3 (Located in Kit Plate 6, well 20D)</li>
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<li>Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)</li>
 
<li>Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)</li>
 
</ul>
 
</ul>
−
 
+
</div>
−
<br>
+
<br><br>
−
 
+
<div class="subtitle" style="color:#05BAC3" ><strong>Strain used</strong></div>
−
<h5>Strain used</h5>
+
<div class="p">
−
<ul>
+
<ul style="list-style:none">
 
<li>Escherichia coli​ strain DH5α</li>
 
<li>Escherichia coli​ strain DH5α</li>
 
<li>Materials</li>
 
<li>Materials</li>
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<li>Pipettes </li>
 
<li>Pipettes </li>
 
</ul>
 
</ul>
−
 
+
</div>
−
<br>
+
<br><br>
−
 
+
<div class="subtitle" style="color:#05BAC3" ><strong>Machines</strong></div>
−
<h5>Machines</h5>
+
<div class="p" >
−
<ul>
+
<ul style="list-style:none">
 
<li>Thermo Multiskan FC</li>
 
<li>Thermo Multiskan FC</li>
 
<li>BERTHOLD TECHNOLOGIES LB940Mith</li>
 
<li>BERTHOLD TECHNOLOGIES LB940Mith</li>
 
</ul>
 
</ul>
 +
</div>
  
−
<br><br><br>
+
</div>
 +
<br><br><br><br>
  
−
<h3>Protocol</h3>
+
<div>
 +
<div class="title" ><strong style="color:#D5292C">Protocol</strong></div>
 +
<div class="p">
 
<ul>
 
<ul>
 
<li><a href="#">Calibration Protocols</a></li>
 
<li><a href="#">Calibration Protocols</a></li>
 
<li><a href="#">Cell measurement protocol</a></li>
 
<li><a href="#">Cell measurement protocol</a></li>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
 +
<br><br><br><br>
  
−
<br><br><br>
+
<div>
 +
<div class="title" ><strong style="color:#05BAC3">Results</strong></div>
 +
<br><br>
  
−
<h3>Results</h3>
+
<div class="subtitle" ><strong style="color:#05BAC3">Sequencing</strong></div>
−
<h5>Sequencing</h5>
+
<div class="p">
 +
<ul style="list-style:none;margin-left:0">
 
<li>Positive Control ：BBa_I20270</li>
 
<li>Positive Control ：BBa_I20270</li>
 
<li>Negative Control ：BBa_R0040</li>
 
<li>Negative Control ：BBa_R0040</li>
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<li>Test Device 5 ：BBa_J364004</li>
 
<li>Test Device 5 ：BBa_J364004</li>
 
<li>Test Device 6 ：BBa_J364005</li>
 
<li>Test Device 6 ：BBa_J364005</li>
−
<p>
+
</ul>
−
<h6>>BBa_I20270 Part-only sequence (919 bp)</h6>
+
</div>
 +
<br><br>
 +
 
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_I20270 Part-only sequence (919 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtccc
 
ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtccc
 
aattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattt
 
aattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattt
Line 94: Line 129:
 
aaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatca
 
aaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatca
 
aataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggt
 
aataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggt
−
gggcctttctgcgtttata
+
gggcctttctgcgtttata<br><br>
−
<h6>>BBa_R0040 Part-only sequence (54 bp)</h6>
+
 
−
tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac
+
</p></div>
−
<h6>>BBa_J364000 Part-only sequence (918 bp)</h6>
+
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_R0040 Part-only sequence (54 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 +
tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac<br><br>
 +
 
 +
</p></div>
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_J364000 Part-only sequence (918 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
tttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca
 
tttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca
 
attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta
 
attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta
Line 107: Line 150:
 
aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa
 
aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa
 
ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg
 
ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg
−
ggcctttctgcgtttata
+
ggcctttctgcgtttata<br><br></p>
−
<h6>>BBa_J364001 Part-only sequence (918 bp)</h6>
+
</div>
 +
 
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_J364001 Part-only sequence (918 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
tttacggctagctcagtcctaggtatagtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca
 
tttacggctagctcagtcctaggtatagtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca
 
attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta
 
attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta
Line 118: Line 165:
 
aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa
 
aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa
 
ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg
 
ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg
−
ggcctttctgcgtttata
+
ggcctttctgcgtttata<br><br></p>
−
<h6>>BBa_J364002 Part-only sequence (918 bp)</h6>
+
</div>
 +
 
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_J364002 Part-only sequence (918 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
ttgacagctagctcagtcctagggattgtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca
 
ttgacagctagctcagtcctagggattgtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca
 
attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta
 
attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta
Line 129: Line 180:
 
aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa
 
aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa
 
ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg
 
ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg
−
ggcctttctgcgtttata
+
ggcctttctgcgtttata<br><br></p>
−
<h6>>BBa_J364003 Part-only sequence (990 bp)</h6>
+
</div>
 +
 
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_J364003 Part-only sequence (990 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
tttacagctagctcagtcctaggtattatgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac
 
tttacagctagctcagtcctaggtattatgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac
 
atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta
 
atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta
Line 140: Line 195:
 
gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt
 
gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt
 
ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact
 
ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact
−
gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
+
gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata<br><br></p>
−
<h6>>BBa_J364004 Part-only sequence (990 bp)</h6>
+
</div>
 +
 
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_J364004 Part-only sequence (990 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
tttacggctagctcagtcctaggtatagtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac
 
tttacggctagctcagtcctaggtatagtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac
 
atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta
 
atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta
Line 151: Line 210:
 
gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt
 
gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt
 
ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact
 
ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact
−
gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
+
gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata<br><br></p>
−
<h6>>BBa_J364005 Part-only sequence (990 bp)</h6>
+
</div>
 +
 
 +
<div class="subtitle" ><strong style="color:#05BAC3">>BBa_J364005 Part-only sequence (990 bp)</strong></div>
 +
<div class="p">
 +
<p style="textindent:0;">
 
ttgacagctagctcagtcctagggattgtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac
 
ttgacagctagctcagtcctagggattgtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac
 
atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta
 
atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta
Line 162: Line 225:
 
gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt
 
gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt
 
ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact
 
ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact
−
gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata</p>
+
gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata<br><br></p>
 +
</div>
 +
</div>
 +
<br><br><br><br>
  
−
<br><br><br>
+
<div>
 +
<div class="title" >
 +
<strong style="color:#D5292C">Data</strong>
 +
</div>
 +
<br><br>
  
−
<h3>Data</h3>
+
<div class="subtitle" style="text-align:center"><strong style="color:#D5292C">OD600 Reference Point</strong></div>
−
<h5>OD600 Reference Point</h5>
+
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/f/fb/Interlab_table1.png" style="width:600px;margin:0 auto"></p>
−
<p>Table 1. OD600 Reference Point.</p>
+
<div class="tu">
−
<img src="">tabel
+
  <p style="text-align:center"><strong>Table 1</strong>. OD600 Reference Point.</p>
 +
  <p>&nbsp;</p>
 +
</div>
 +
<br><br>
  
−
<h5>Fluorescein Standard Curve</h5>
+
<div class="subtitle"  style="text-align:center"><strong style="color:#D5292C">Fluorescein Standard Curve</strong></div>
−
<p>Table 2. Data of Fluorescein Standard Curve.</p>
+
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/5/5d/Interlab_table2.png" style="width:100%;margin:0 auto"></p>
−
<img src="">tabel
+
<div class="tu"  style="text-align:center"><strong>Table 2. </strong>Data of Fluorescein Standard Curve.</div>
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/6/6b/Interlab_figure1.png" style="width:400px;margin:0 auto"></p>
 +
<div class="tu"  style="text-align:center"><strong>Figure 1</strong></div>
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/0/00/Interlab_figure2.png" style="width:400px;margin:0 auto"></p>
 +
<div class="tu"  style="text-align:center">
 +
  <p  style="text-align:center" ><strong>Figure 2</strong></p>
 +
  <p><br><br>
 +
</p></div>
 +
<div class="subtitle" style="text-align:center" ><strong style="color:#D5292C">Normalization</strong></div>
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/1/1c/Interlab_table3.png"width="600px"></p>
 +
<div class="tu">
 +
  <p  style="text-align:center"><strong>Table 3.</strong> Normalization.</p>
 +
  <p>&nbsp;</p>
 +
</div>
 +
<div class="subtitle"  style="text-align:center" ><strong style="color:#D5292C">Cell Measurement</strong></div>
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/d/da/Interlab_table4.png" width="100%"></p>
 +
<p></p>
 +
<div class="tu"  style="text-align:center"><strong>Table 4.</strong> Raw data of OD600 measurement.</div>
  
−
<h5>Normalization</h5>
+
  <p>&nbsp;</p>
−
<p>Table 1. OD600 Reference Point.Table 3. Normalization.</p>
+
</div>
−
<img src="">tabel5
+
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/c/c7/Interlab_table5.png"width="100%"></p>
 +
<div class="tu" style="text-align:center">
 +
  <p  style="text-align:center"><strong>Table 5. </strong>Blank subtraction and correction of OD600 measurement.</p>
 +
  <p>&nbsp;</p>
 +
</div>
  
−
<h5>Cell Measurement</h5>
+
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/b/b5/Interlab_figure3.png"width="400px"></p>
−
<p>Table 4. Raw data of OD600 measurement.</p>
+
<div class="tu"  style="text-align:center">
−
<img src="">tabel
+
  <p style="text-align: center"><strong>Figure 3.</strong> Blank subtraction and correction of Abs600 measurement.</p>
 +
</div>
  
  
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/8/87/Interlab_table6.png"width="100%"></p><div class="tu">
 +
  <p  style="text-align:center"><strong>Table 6.</strong> Raw data of fluorescence measurement.</p>
 +
  <p>&nbsp;</p>
 +
</div>
  
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/b/bc/Interlab_table7.png"width="100%"></p><div class="tu">
 +
  <p  style="text-align:center"><strong>Table 7.</strong> Blank substraction and correction of fluorescence measurement.</p>
 +
  <p>&nbsp;</p>
 +
</div>
  
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/2/20/Interlab_figure4.png"width="400px"></p><div class="tu">
 +
  <p  style="text-align:center"><strong>Figure 4.</strong> Blank subtraction and correction of fluorescence measurement.</p>
 +
  <p>&nbsp;</p>
 +
</div>
  
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/7/7b/Interlab_table8.png"width="100%"></p><div class="tu">
 +
  <p  style="text-align:center"><strong>Table 8.</strong>Raw data of Fl/Abs600.</p>
 +
  <p>&nbsp;</p>
 +
</div>
 +
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/2/2e/Interlab_table9.png"width="600px"></p><div class="tu">
 +
  <p  style="text-align:center"><strong>Table 9.</strong> Raw data of average</p>
 +
  <p>&nbsp;</p>
 +
</div>
 +
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/1/17/Interlab_table10.png"width="600px"></p ><div class="tu"  style="text-align:center"><strong>Table 10.</strong> Raw data of SD</div>
 +
 +
 +
<p style="text-align: center"><img src="https://static.igem.org/mediawiki/2017/2/24/Interlab_figure5.png"width="400px"></p><div class="tu"  style="text-align:center"><strong>Figure 5. </strong>Average level of devices.</div>
 +
<p></p>
 +
 +
 +
 +
 +
</div>
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</div>
 
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Latest revision as of 09:02, 8 October 2017



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 be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.




Design

 

We often used fluorescence as an indicator of promoter activity. However, the ability to repeat measurements in different labs has been difficult. To solve this problem, InterLab study has been developed as a robust measurement procedure for green fluorescent protein (GFP) which is one of the most used markers in synthetic biology.
This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.




Materials


Plasmids used
  • Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)
  • Negative Control Device: R0040 in pSB1C3 (Located in Kit Plate 6, well 20D)
  • Test Device 1: J23101+I13504 in pSB1C3 (Located in Kit Plate 6, well 20F)
  • Test Device 2: J23106+I13504 in pSB1C3 (Located in Kit Plate 6, well 20H)
  • Test Device 3: J23117+I13504 in pSB1C3 (Located in Kit Plate 6, well 20J)
  • Test Device 4: J23101.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20L)
  • Test Device 5: J23106.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20N)
  • Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)


Strain used
  • Escherichia coli​ strain DH5α
  • Materials
  • 1ml LUDOX
  • H​2​0
  • 96 well plate
  • fluorescein
  • 10ml 1xPBS
  • LB (Luria Bertani) media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
  • 50 ml Falcon tube
  • Incubator at 37°C
  • 1.5 ml eppendorf tubes
  • Ice bucket with ice
  • Pipettes


Machines
  • Thermo Multiskan FC
  • BERTHOLD TECHNOLOGIES LB940Mith








Results


Sequencing
  • Positive Control ：BBa_I20270
  • Negative Control ：BBa_R0040
  • Test Device 1 ：BBa_J364000
  • Test Device 2 ：BBa_J364001
  • Test Device 3 ：BBa_J364002
  • Test Device 4 ：BBa_J364003
  • Test Device 5 ：BBa_J364004
  • Test Device 6 ：BBa_J364005


>BBa_I20270 Part-only sequence (919 bp)

ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtccc aattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattt atttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaac agcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaa gtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataac tcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcag accattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacga aaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatca aataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggt gggcctttctgcgtttata

>BBa_R0040 Part-only sequence (54 bp)

tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac

>BBa_J364000 Part-only sequence (918 bp)

tttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta tttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaaca gcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaag tttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcaga ccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaa aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg ggcctttctgcgtttata

>BBa_J364001 Part-only sequence (918 bp)

tttacggctagctcagtcctaggtatagtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta tttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaaca gcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaag tttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcaga ccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaa aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg ggcctttctgcgtttata

>BBa_J364002 Part-only sequence (918 bp)

ttgacagctagctcagtcctagggattgtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtccca attcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaattta tttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaaca gcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaag tttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataact cacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcaga ccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaa aagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaa ataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtg ggcctttctgcgtttata

>BBa_J364003 Part-only sequence (990 bp)

tttacagctagctcagtcctaggtattatgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta atgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttcc atggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgccc gaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaa tcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaa acaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

>BBa_J364004 Part-only sequence (990 bp)

tttacggctagctcagtcctaggtatagtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta atgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttcc atggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgccc gaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaa tcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaa acaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

>BBa_J364005 Part-only sequence (990 bp)

ttgacagctagctcagtcctagggattgtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaac atcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtta atgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttcc atggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgccc gaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaa tcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaa acaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagt ttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagact gggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata





Data


OD600 Reference Point

Table 1. OD600 Reference Point.

 



Fluorescein Standard Curve

Table 2. Data of Fluorescein Standard Curve.

Figure 1

Figure 2



Normalization

Table 3. Normalization.

 

Cell Measurement

Table 4. Raw data of OD600 measurement.

 

Table 5. Blank subtraction and correction of OD600 measurement.

 

Figure 3. Blank subtraction and correction of Abs600 measurement.

Table 6. Raw data of fluorescence measurement.

 

Table 7. Blank substraction and correction of fluorescence measurement.

 

Figure 4. Blank subtraction and correction of fluorescence measurement.

 

Table 8.Raw data of Fl/Abs600.

 

Table 9. Raw data of average

 

Table 10. Raw data of SD

Figure 5. Average level of devices.