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

 
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<h1><font size="6" color="black">InterLab</font></h1>
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<font size="5" color="red">Introduction</font> <br>
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        <title>Royal Hotel</title>
We took part in the Fifth International InterLab Measurement Study which ains to achieve the purpose of comparative measurement. The goal of this study is to obtain large amounts of data from labs across the world,to develop absolute units for measurements GFP in a plate reader to eliminate variation between labs. <br>
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<font size="5" color="red">Materials</font> <br>
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Plate reader: Synergy H1 (Biotek) <br>
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Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom) <br>
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Cell culture shaker: ZWYR-200D <br>
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<br>
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Devices: <br>  
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Negative control :BBa_R0040 <br>
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        <!--================Header Area =================-->
Positive control :BBa_I20270 <br>
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Device 1: BBa_J364000 <br>
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Device 2: BBa_J364001 <br>
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Device 3: BBa_J364002 <br>
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Device 4: BBa_J364007 <br>
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Device 5: BBa_J364008 <br>
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Note: for Device 5, we have not transformed it into DH5⍺ competent cells successfully for many times, therefore, we thank IGEM team of Nanjing University for providing the Device 5.<br>
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        <br> <br> <br> <br> <br> <br>
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        <div align="center" class="section4"><img src="https://static.igem.org/mediawiki/2018/f/fc/T--AHUT_China--_interlab1562.jpg" width="602"  alt=""/></div>
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              <div class="section_title ">
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                <div align="center"><div align="center"><h2>Interlab</h2></div>
 +
<hr>
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<h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Introduction</h2></div><hr>
 +
                  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >We took part in the Fifth International InterLab Measurement Study which ains to achieve the purpose of comparative measurement. The goal of this study is to obtain large amounts of data from labs across the world,to develop absolute units for measurements GFP in a plate reader to eliminate variation between labs.</p><br><br><br><br>
 +
<div align="center"><h2 class="title_color">Materials</h2></div><hr>
 +
                  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >Plate reader: Synergy H1 (Biotek)<br>
 +
Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)<br>
 +
Cell culture shaker: ZWYR-200D<br><br></p>
 +
<p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >Devices:<br>
 +
Negative control :<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a> <br>
 +
Positive control :<a href="http://parts.igem.org/Part:BBa_I20270">BBa_I20270</a> <br>  
 +
Device 1:<a href="
 +
http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a><br>
 +
Device 2:<a href="http://parts.igem.org/Part:BBa_J364001">BBa_J364001</a><br>
 +
Device 3:<a href="
 +
http://parts.igem.org/Part:BBa_J364002">BBa_J364002  </a> <br>
 +
Device 4:<a href="http://parts.igem.org/Part:BBa_J364007">BBa_J364007  </a> <br>
 +
Device 5:<a href="http://parts.igem.org/Part:BBa_J364008">BBa_J364008  </a> <br>
 +
Device 6:<a href="http://parts.igem.org/Part:BBa_J364009">BBa_J364009  </a><br></p>
 +
<p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >Note: for Device 5, we have not transformed it into DH5⍺ competent cells successfully for many times, therefore, we thank IGEM team of Nanjing University for providing the Device 5.<br>
 
Calibration material: Provided in the 2018 IGEM distribution kit <br>
 
Calibration material: Provided in the 2018 IGEM distribution kit <br>
<br>
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Microorganism: Escherichia coli DH5⍺ strains<br>
Microorganism: Escherichia coli DH5⍺ strains <br>
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</p><br><br><br><br>
 
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<div align="center"><h2 class="title_color">Methods</h2></div><hr>
 
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                  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >Following iGEM requirements, Team AHUT_China performed measurements according to these 2018 InterLab Protocols <a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf</a> </p><br><br><br><br>
 
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<div align="center"><h2 class="title_color">Results</h2></div><hr>
 
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                  <h3>1.OD 600 reference point</h3> <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >
<font size="5" color="red">Methods</font> <br>
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Using OD 600 and H<span style="font-size: 13px">2</span>O to generate the conversion factor for the transformation later. The average of OD600 is 0.063; the correction factor (OD600/ABS600) is 3.500
Following iGEM requirements, Team AHUT_China performed measurements according to these 2018 InterLab Protocols  
+
</p><br><br><br>
<a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf" target="_blank">https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf</a><br>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/c/cb/T--AHUT_China--_LUDOX_correct_result.jpg" width="650" alt=""/></div><br>
 
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<p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 1 LUDOX correct value</p>
 
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<br><br><br>
 
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  <h3>2.Particle standard curve</h3>
 
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                  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >
<font size="5" color="red">Results</font> <br>
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We obtained the two Particle Standard Curve (normal and log scale).
1. OD 600 reference point <br>
+
</p>
Using OD 600 and H2O to generate the conversion factor for the transformation later. The average of OD600 is 0.063; the correction factor (OD600/ABS600) is 3.500  
+
<br><br><br>
 
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/6/65/T--AHUT_China--_Fig._2_Particle_Standard_Curve.jpg" width="800alt=""/></div><p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 2 Particle Standard Curve</p>
 
+
<br><br><br>
 
+
<div align="center"><img src="https://static.igem.org/mediawiki/2018/7/7e/T--AHUT_China--_Fig._3_Particle_Standard_Curve_%28log_scale%29.jpg" width="800alt=""/></div><p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 3 Particle Standard Curve (log scale)</p>
<img src="https://static.igem.org/mediawiki/2018/3/35/T--AHUT_China--result.png" width="233" height="166" >
+
<br><br><br>
 
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<h3>3.Fluorescein standard curve</h3> <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >
<p style="color:red;font-size:15px;text-align:center;">Fig. 1 LUDOX correct value</p> <br>
+
Dilution serious of fluorescein were prepared and measured in a 96 well plate. A standard curve is generated to correct the cell based readings to an equivalent fluorescein concentration.We obtained the two Fluorescein Standard Curve (normal and log scale).
<br>
+
</p>
</p>
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<br><br><br>
 
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<p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/24/T--AHUT_China--_Fig._4_Fluorescein_Standard_Curve.jpg" width="800alt=""/></div><p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 4 Fluorescein Standard Curve</p>
2. Particle standard curve <br>
+
<br><br><br>
We obtained the two Particle Standard Curve (normal and log scale). <br>
+
<div align="center"><img src="https://static.igem.org/mediawiki/2018/a/a9/T--AHUT_China--_Fig._5_Fluorescein_Standard_Curve_%28log_scale%29.jpg" width="800" alt=""/></div><p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 5 Fluorescein Standard Curve (log scale)</p>
 
+
<br><br><br>
<img src="https://static.igem.org/mediawiki/2018/2/25/T--AHUT_China--Fig._2_Particle_Standard_Curve.png" width="218pxheight="300px" align="middle" />
+
<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 2 Particle Standard Curve</p> <br>
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  <h3>4.Cell measurements</h3>
 
+
  </ol>
<img src="https://static.igem.org/mediawiki/2018/d/da/T--AHUT_China--Fig._3_Particle_Standard_Curve_%28log_scale%29.png" width="218pxheight="300px" align="middle" />
+
<p></p><br><br><br>
<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 3 Particle Standard Curve (log scale) </p> <br>
+
 
</p>
+
<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/21/T--AHUT_China--_Fig._6_Fluorescence_Measurements_Curve_.jpg" width="800alt=""/></div><p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 6 Fluorescence Measurements Curve</p>
 
+
<br><br><br>
<p>
+
    <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >Test devices 1 and 4 show high fluorescence intensity. Test devices 2 show a modest fluorescence intensity alone with positive control group, while devices3,5,6 barely show low fluorescence intensity alone with the negative control group.
3. Fluorescein standard curve <br>
+
</p><br><br><br>
Dilution serious of fluorescein were prepared and measured in a 96 well plate. A standard curve is generated to correct the cell based readings to an equivalent fluorescein concentration.
+
 
We obtained the two Fluorescein Standard Curve (normal and log scale). <br>
+
<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/36/T--AHUT_China--_Fig._7_Raw_OD600_Curve_.jpg" width="800alt=""/></div><br><div align="center">
+
  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 7 Raw OD600 Curve</p>
<img src="https://static.igem.org/mediawiki/2018/2/28/T--AHUT_China--Fig._4_Fluorescein_Standard_Curve.png" width="218pxheight="300px" align="middle" />
+
<br><br><br>
<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 4 Fluorescein Standard Curve</p> <br>
+
    <h3>5.We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures</h3>
 
+
<br><br><br>  
<img src="https://static.igem.org/mediawiki/2018/a/ae/T--AHUT_China--Fig._5_Fluorescein_Standard_Curve_%28log_scale%29.png" width="218px" height="300px" align="middle" />
+
<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f1/T--AHUT_China--_Fig._8_CFU_Result.jpg" width="800alt=""/></div>
<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 5 Fluorescein Standard Curve (log scale)</p> <br>
+
<br><br>
</p>
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    <div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e5/T--AHUT_China--_Fig._8_CFU_Result1.jpg" width="800alt=""/></div>
 
+
  <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 14px;text-align: center;">Fig. 8 CFU Result</p>
<p>
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  <p ></p>
4. Cell measurements <br>
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<br><br><br><br>
 
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<div align="center"><h2 class="title_color">Discussion</h2></div>
<img src="https://static.igem.org/mediawiki/2018/8/89/T--AHUT_China--Fig._6_Fluorescence_Measurements_Curve.png" width="218height="300" />
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                <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >For Figure 3, the log graph isn’t a straight line but not 1:1 slope. In figure 6, highest fluorescence was obtained from device 4, closely followed by test device 1. Test device 2 and positive control group show a modest fluorescence intensity and device 5,6 show low fluorescence intensity, while test devices 3 barely have any fluorescence signal as well as the negative group.  
<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 6 Fluorescence Measurements Curve</p> <br>
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<br><br><br><br>         
<p>
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<div align="center"><h2 class="title_color">Conclusion</h2></div>
Test devices 1 and 4 show high fluorescence intensity. Test devices 2 show a modest fluorescence intensity alone with positive control group, while devices3,5,6 barely show low fluorescence intensity alone with the negative control group. <br>
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                <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px;" >In the case of complying laboratory safety guidelines , we completed the protocol and achieved the expected results.We completed the interlab form, which proved that we made contributions to the interlab study.
</p>
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<img src="https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--Fig._7_Raw_OD600_Curve.png" width="218pxheight="300px" align="middle" />
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<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 7 Raw OD600 Curve</p> <br>
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<p>
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Show the growing curve of different cells indirectlly. All test devices show a high growing. Comparing to the positive control, the negative control group shows a high growing.<br>
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</p>
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<p>
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5. We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures <br>
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<img src="https://static.igem.org/mediawiki/2018/f/fb/T--AHUT_China--Fig._8_CFU_Result1.png" width="218pxheight="300px" align="middle" /> <br>
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<img src="https://static.igem.org/mediawiki/2018/c/ce/T--AHUT_China--Fig._8_CFU_Result2.png" width="218pxheight="300px" align="middle" /> <br>
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<p style="font-family:arial;color:red;font-size:15px;text-align:center;">Fig. 8 CFU Result</p> <br>
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<font size="5" color="red">Discussion</font> <br>
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<p>
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For Figure 3, the log graph isn’t a straight line but not 1:1 slope. In figure 6, highest fluorescence was obtained from device 4, closely followed by test device 1. Test device 2 and positive control group show a modest fluorescence intensity and device 5,6 show low fluorescence intensity, while test devices 3 barely have any fluorescence signal as well as the negative group.  <br>
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</p>
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<font size="5" color="red">Conclusion</font> <br>
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<p>
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It was certainly a technical challenge to Participate in the InterLab Study. Performing the prescribed protocols with adherence to all the InterLab guidelines yielded parts of expected results, and with the completed InterLab Google Forms, confirms our team participation in this InterLab Study. <br>
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Latest revision as of 03:03, 18 October 2018

Royal Hotel Royal Hotel







Interlab


     Introduction


We took part in the Fifth International InterLab Measurement Study which ains to achieve the purpose of comparative measurement. The goal of this study is to obtain large amounts of data from labs across the world,to develop absolute units for measurements GFP in a plate reader to eliminate variation between labs.





Materials


Plate reader: Synergy H1 (Biotek)
Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)
Cell culture shaker: ZWYR-200D

Devices:
Negative control :BBa_R0040
Positive control :BBa_I20270
Device 1:BBa_J364000
Device 2:BBa_J364001
Device 3:BBa_J364002
Device 4:BBa_J364007
Device 5:BBa_J364008
Device 6:BBa_J364009

Note: for Device 5, we have not transformed it into DH5⍺ competent cells successfully for many times, therefore, we thank IGEM team of Nanjing University for providing the Device 5.
Calibration material: Provided in the 2018 IGEM distribution kit
Microorganism: Escherichia coli DH5⍺ strains





Methods


Following iGEM requirements, Team AHUT_China performed measurements according to these 2018 InterLab Protocols https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf





Results


1.OD 600 reference point

Using OD 600 and H2O to generate the conversion factor for the transformation later. The average of OD600 is 0.063; the correction factor (OD600/ABS600) is 3.500





Fig. 1 LUDOX correct value




2.Particle standard curve

We obtained the two Particle Standard Curve (normal and log scale).




Fig. 2 Particle Standard Curve




Fig. 3 Particle Standard Curve (log scale)




3.Fluorescein standard curve

Dilution serious of fluorescein were prepared and measured in a 96 well plate. A standard curve is generated to correct the cell based readings to an equivalent fluorescein concentration.We obtained the two Fluorescein Standard Curve (normal and log scale).




Fig. 4 Fluorescein Standard Curve




Fig. 5 Fluorescein Standard Curve (log scale)




4.Cell measurements




Fig. 6 Fluorescence Measurements Curve




Test devices 1 and 4 show high fluorescence intensity. Test devices 2 show a modest fluorescence intensity alone with positive control group, while devices3,5,6 barely show low fluorescence intensity alone with the negative control group.





Fig. 7 Raw OD600 Curve




5.We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures






Fig. 8 CFU Result





Discussion

For Figure 3, the log graph isn’t a straight line but not 1:1 slope. In figure 6, highest fluorescence was obtained from device 4, closely followed by test device 1. Test device 2 and positive control group show a modest fluorescence intensity and device 5,6 show low fluorescence intensity, while test devices 3 barely have any fluorescence signal as well as the negative group.





Conclusion

In the case of complying laboratory safety guidelines , we completed the protocol and achieved the expected results.We completed the interlab form, which proved that we made contributions to the interlab study.