Difference between revisions of "Team:SHSBNU China/InterLab"

 
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<p style="font-family: Serif;font-size: 3.5vw; font-weight: bold;text-align: center;margin: 0.5vw 0vw 0.5vw 0vw">Sample</p>
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<h6 id="menu_intro">InterLab</h6>
 
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<a class="snd_class" href="">SHSBNU_China</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#Background">Background</a>
 
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<a class="trd_class" href="">classfication</a>
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<div class="second_classfication">
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#Design">Design</a>
 
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<a class="snd_class" href="">SHSBNU_China</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#MM">Material and Methods</a>
 
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<a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#PU">Plasmids Used</a>
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<div class="third_classfication">
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<a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#SU">Materials</a>
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<div class="third_classfication">
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<a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#Machines">Machines</a>
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</div>
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<div class="third_classfication">
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<a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#Software">Software</a>
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<div class="third_classfication">
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<a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/InterLab#Results">Results</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Experiments">Experiment</a>
 
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<h2>I. Background</h2>
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<h2 id="Background">I. Background</h2>
 
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“All of 2018 iGEM teams are invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which is aimed to figure out and correct the sources of systematic variability in synthetic biology measurements. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.
+
“All of 2018 iGEM teams were invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which was aimed to figure out and correct the sources of systematic variability in synthetic biology measurements. 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>
 
</p>
 
</div>
 
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<h2>II. Design</h2>
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<h2 id="Design">II. Design</h2>
 
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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 a real-time monitor without disrupting cells.
+
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 a real-time monitor without disrupting cells.
 
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<h2>III. Material and Methods</h2>
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<h2 id="MM">III. Material and Methods</h2>
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<h3>a. Plasmids Used</h3>
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<h3 id="PU">a. Plasmids Used</h3>
 
<div class="content">
 
<div class="content">
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<li class="content_list"><p>Negative control  BBa_R0040      (provided in kit plate)</p></li>
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<table bgcolor="#f0f0f0" cellspacing="1px">
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<li class="content_list"><p>Positive control  BBa_I20270    (provided in kit plate)</p></li>
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<li class="content_list"><p>Test Device 1      BBa_J364000    (provided in kit plate)</p></li>
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<li class="content_list"><p>Test Device 2      BBa_J364001    (provided in kit plate)</p></li>
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<li class="content_list"><p>Test Device 3      BBa_J36400    (provided in kit plate)</p></li>
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<li class="content_list"><p>Test Device 4      BBa_J364007    (provided in kit plate)</p></li>
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<li class="content_list"><p>Test Device 5      BBa_J364008    (provided in kit plate)</p></li>
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<li class="content_list"><p>Test Device 6      BBa_J364009    (provided in kit plate)</p></li>
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−
</div>
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−
<h3>b. Strain used</h3>
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−
<h3>c. Machines</h3>
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−
<h3>d. Software</h3>
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<h3>f. Results</h3>
+
  
 +
<tr bgcolor="#f0f0f0">
 +
<td>Negative control</td>
 +
<td>BBa_R0040</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
  
 +
<tr bgcolor="#f0f0f0">
 +
<td>Positive control</td>
 +
<td>BBa_I20270</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
  
 +
<tr bgcolor="#f0f0f0">
 +
<td>Test Device 1</td>
 +
<td>BBa_J364000</td>
 +
<td>(provided in kit plate)</td>
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</tr>
 +
 +
<tr bgcolor="#f0f0f0">
 +
<td>Test Device 2</td>
 +
<td>BBa_J364001</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
 +
 +
<tr bgcolor="#f0f0f0">
 +
<td>Test Device 3</td>
 +
<td>BBa_J36400</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
 +
 +
<tr bgcolor="#f0f0f0">
 +
<td>Test Device 4</td>
 +
<td>BBa_J364007</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
 +
 +
<tr bgcolor="#f0f0f0">
 +
<td>Test Device 5</td>
 +
<td>BBa_J364008</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
 +
 +
<tr bgcolor="#f0f0f0">
 +
<td>Test Device 6</td>
 +
<td>BBa_J364009</td>
 +
<td>(provided in kit plate)</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h3 id="SU">b. Materials</h3>
 
<div class="content">
 
<div class="content">
−
<div style="float:left" class="content_pic_left">
+
<li class="content_list">Competent cells (Escherichia coli strain DH5α)</li>
−
<img class="pictures" id = "" src=""/>
+
<li class="content_list">1x PBS</li>
−
<p class="pic_text">This is a Picture. This is a Picture. This is a Picture. This is a Picture.</p>
+
<li class="content_list">ddH2O</li>
 +
<li class="content_list">LB media</li>
 +
<li class="content_list">Chloramphenicol</li>
 +
<li class="content_list">50 ml Falcon tube</li>
 +
<li class="content_list">1.5 ml Eppendorf tubes</li>
 +
<li class="content_list">Ice bucket with ice</li>
 +
<li class="content_list">Micropipettes</li>
 +
<li class="content_list">Micropipette tips</li>
 +
<li class="content_list">96 well plates, black with clear flat bottom preferred</li>
 +
<li class="content_list">LUDOX CL-X (provided in kit)</li>
 +
<li class="content_list">Fluorescein sodium salt (provided in kit)</li>
 +
<li class="content_list">Silica Beads (Provided in kit)</li>
 +
</div>
 +
<h3 id="Machines">c. Machines</h3>
 +
<div class="content">
 +
<p class="text">
 +
Spectrophotometer: Thermo Scientific Varioskan Flash
 +
</p>
 +
</div>
 +
<h3 id="Software">d. Software</h3>
 +
<div class="content">
 +
<p class="text">
 +
Microsoft Excel 2016
 +
</p>
 +
</div>
 +
<h3 id="Results">e. Results</h3>
 +
<h5 class="forth_classification">
 +
OD600 Reference Point
 +
</h5>
 +
<div class="content">
 +
<div style="width: 46vw;" class="content_pic_left">
 +
<img class="pictures" id = "29400" src="https://static.igem.org/mediawiki/2018/8/8f/T--SHSBNU_China--29400.png"/>
 +
<p class="pic_text">Table 1. OD600 Reference Point.</p>
 +
</div>
 +
</div>
 +
<h5 class="forth_classification">
 +
Fluorescein Standard Curve
 +
</h5>
 +
<div class="content">
 +
<div style="width: 46vw;" class="content_pic_left">
 +
<img class="pictures" id = "29401" src="https://static.igem.org/mediawiki/2018/5/56/T--SHSBNU_China--29401.png"/>
 +
<p class="pic_text">Table2. Original data of Fluorescein standard curve</p>
 +
<img class="pictures" id = "29402" src="https://static.igem.org/mediawiki/2018/7/71/T--SHSBNU_China--29402.png"/>
 +
<p class="pic_text">Figure1. Fluorescein Standard Curve</p>
 +
</div>
 +
</div>
 +
<h5 class="forth_classification">
 +
Particle Standard Curve
 +
</h5>
 +
<div class="content">
 +
<div style="width: 46vw;" class="content_pic_left">
 +
<img class="pictures" id = "29404" src="https://static.igem.org/mediawiki/2018/2/2a/T--SHSBNU_China--29404.png"/>
 +
<p class="pic_text">Figure 2. Particle Standard Curve.</p>
 +
<img class="pictures" id = "29403" src="https://static.igem.org/mediawiki/2018/a/af/T--SHSBNU_China--29403.png"/>
 +
<p class="pic_text">Table 3. Original data of Particle Standard Curve</p>
 +
<img class="pictures" id = "29405" src="https://static.igem.org/mediawiki/2018/8/8c/T--SHSBNU_China--29405.png"/>
 +
<p class="pic_text">Figure 3. Particle Standard Curve (log scale).</p>
 +
</div>
 +
</div>
 +
<h5 class="forth_classification">
 +
Cell measurement
 +
</h5>
 +
<div class="content">
 +
<div style="width: 46vw;" class="content_pic_left">
 +
<img class="pictures" id = "29406" src="https://static.igem.org/mediawiki/2018/8/8c/T--SHSBNU_China--29406.png"/>
 +
<p class="pic_text">Table 4. Fluorescence Intensity of E.coli transformed with Device 1 to 6 and two control groups. Intensities was measured under OD600 using Plate Reader for 6 hours.</p>
 
</div>
 
</div>
 
<p class="text">
 
<p class="text">
−
This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. 
+
As shown in Table.4, the combination of RBS and promoter in Device exhibited the strongest fluorescent intensity in 6h. Among these six devices, Device 3 almost had no fluorescent intensity.  
 
</p>
 
</p>
−
<div style="float:" class="content_pic_right">
+
</div>
−
<img class="pictures" id = "" src=""/>
+
<h5 class="forth_classification">
−
<p class="pic_text">This is a Picture. This is a Picture. This is a Picture. This is a Picture.</p>
+
Original data link:
 +
</h5>
 +
<div class="content">
 +
<div style="width: 46vw;" class="content_pic_left">
 +
<img class="pictures" id = "29407" src="https://static.igem.org/mediawiki/2018/2/24/T--SHSBNU_China--29407.png"/>
 +
<p class="pic_text">Table 5. Bacterial concentration of E.coli transformed with Device1-6, Negative Control & Positive Control. Intensities were measured under OD600 using plate for 6 hours. </p>
 
</div>
 
</div>
 
<p class="text">
 
<p class="text">
−
This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture. This is a Picture.
+
As shown in Table.5, except Device 1, the concentrations of E.coli transformed with another five devices were almost the same.  
 +
</p>
 +
</div>
 +
<h5 class="forth_classification">
 +
Normalizing
 +
</h5>
 +
<div class="content">
 +
<p class="text">
 +
Counting colony-forming units
 +
</p>
 +
<div style="width: 46vw;" class="content_pic_left">
 +
<img class="pictures" id = "29408" src="https://static.igem.org/mediawiki/2018/6/67/T--SHSBNU_China--29408.png"/>
 +
<p class="pic_text">Figure 5. Counted by Promega Colony Counter, bacterial concentration of E.coli transformed with Positive Control & Negative Control. </p>
 +
</div>
 +
<p class="text">
 +
As shown in Figure.5, bacterial colonies were counted in order to determine whether absolute cell count or colony-forming units (CFUs) can reduce lab-to-lab variability in fluorescence measurements.  
 +
</p>
 +
<p class="text">
 +
It is evident that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3. Additionally, CFUs could not replace the lab measurement because bacterial colonies was so crowded that it was impossible to count precisely by Promega Colony Counter.  
 
</p>
 
</p>
 
</div>
 
</div>

Latest revision as of 00:42, 18 October 2018

Section Sample

InterLab

I. Background

“All of 2018 iGEM teams were invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which was aimed to figure out and correct the sources of systematic variability in synthetic biology measurements. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.

II. Design

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 a real-time monitor without disrupting cells.

Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.

III. Material and Methods

a. Plasmids Used

Negative control BBa_R0040 (provided in kit plate)
Positive control BBa_I20270 (provided in kit plate)
Test Device 1 BBa_J364000 (provided in kit plate)
Test Device 2 BBa_J364001 (provided in kit plate)
Test Device 3 BBa_J36400 (provided in kit plate)
Test Device 4 BBa_J364007 (provided in kit plate)
Test Device 5 BBa_J364008 (provided in kit plate)
Test Device 6 BBa_J364009 (provided in kit plate)

b. Materials

  • Competent cells (Escherichia coli strain DH5α)
  • 1x PBS
  • ddH2O
  • LB media
  • Chloramphenicol
  • 50 ml Falcon tube
  • 1.5 ml Eppendorf tubes
  • Ice bucket with ice
  • Micropipettes
  • Micropipette tips
  • 96 well plates, black with clear flat bottom preferred
  • LUDOX CL-X (provided in kit)
  • Fluorescein sodium salt (provided in kit)
  • Silica Beads (Provided in kit)

    • c. Machines

    Spectrophotometer: Thermo Scientific Varioskan Flash

    d. Software

    Microsoft Excel 2016

    e. Results

    OD600 Reference Point

    Table 1. OD600 Reference Point.

    Fluorescein Standard Curve

    Table2. Original data of Fluorescein standard curve

    Figure1. Fluorescein Standard Curve

    Particle Standard Curve

    Figure 2. Particle Standard Curve.

    Table 3. Original data of Particle Standard Curve

    Figure 3. Particle Standard Curve (log scale).

    Cell measurement

    Table 4. Fluorescence Intensity of E.coli transformed with Device 1 to 6 and two control groups. Intensities was measured under OD600 using Plate Reader for 6 hours.

    As shown in Table.4, the combination of RBS and promoter in Device exhibited the strongest fluorescent intensity in 6h. Among these six devices, Device 3 almost had no fluorescent intensity.

    Original data link:

    Table 5. Bacterial concentration of E.coli transformed with Device1-6, Negative Control & Positive Control. Intensities were measured under OD600 using plate for 6 hours.

    As shown in Table.5, except Device 1, the concentrations of E.coli transformed with another five devices were almost the same.

    Normalizing

    Counting colony-forming units

    Figure 5. Counted by Promega Colony Counter, bacterial concentration of E.coli transformed with Positive Control & Negative Control.

    As shown in Figure.5, bacterial colonies were counted in order to determine whether absolute cell count or colony-forming units (CFUs) can reduce lab-to-lab variability in fluorescence measurements.

    It is evident that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3. Additionally, CFUs could not replace the lab measurement because bacterial colonies was so crowded that it was impossible to count precisely by Promega Colony Counter.