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

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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                <div align="center"> <h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Introduction</h2></div>
 +
                  <p>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>
 +
<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Materials</h2></div>
 +
                  <p>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>
 +
Devices:<br>
 +
Negative control ：BBa_R0040 <br>
 +
Positive control ：BBa_I20270 <br>
 +
Device 1： BBa_J364000  <br>
 +
Device 2： BBa_J364001  <br>
 +
Device 3： BBa_J364002  <br>
 +
Device 4： BBa_J364007  <br>
 +
Device 5： BBa_J364008  <br>
 +
Device 6： BBa_J364009  <br>
 +
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>
 +
Microorganism: Escherichia coli DH5⍺ strains<br>
 +
</p><br>
 +
<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Methods</h2></div>
 +
                  <p>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>
 +
<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Results</h2></div>
 +
                  <h4>1.OD 600 reference point</h4><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
 +
</p><br>
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2018/c/cb/T--AHUT_China--_LUDOX_correct_result.jpg" width="317" height="234" alt=""/></div><br><div align="center">Fig. 1 LUDOX correct value
 +
  </div>
 +
  <h4>2.Particle standard curve</h4>
 +
                  <p>
 +
We obtained the two Particle Standard Curve (normal and log scale).
 +
</p><br>
 +
  <div align="center"><img src="https://static.igem.org/mediawiki/2018/6/65/T--AHUT_China--_Fig._2_Particle_Standard_Curve.jpg" width="701" height="440" alt=""/></div><br><div align="center">Fig. 2 Particle Standard Curve
 +
  </div>
 +
<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="701" height="440" alt=""/></div><br><div align="center">
 +
  Fig. 3 Particle Standard Curve (log scale)
 +
</div>
 +
<h4>3.Fluorescein standard curve</h4><p>
 +
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.<br>
 +
We obtained the two Fluorescein Standard Curve (normal and log scale).
 +
</p><br>
 +
 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/24/T--AHUT_China--_Fig._4_Fluorescein_Standard_Curve.jpg" width="701" height="440" alt=""/></div><br><div align="center">
 +
  Fig. 4 Fluorescein Standard Curve
 +
</div>
 +
<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="701" height="440" alt=""/></div><br><div align="center">
 +
  <div align="center" >Fig. 5 Fluorescein Standard Curve (log scale) </div>
 +
</div>
 +
 +
  <h4>4.Cell measurements</h4>
 +
  </ol>
 +
<p>&nbsp;</p><br>
 +
 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/21/T--AHUT_China--_Fig._6_Fluorescence_Measurements_Curve_.jpg" width="732" height="492" alt=""/></div><br><div align="center">
 +
  <div align="center">Fig. 6 Fluorescence Measurements Curve</div>
 +
</div>
 +
    <p>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.
 +
</p><br>
 +
 
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/36/T--AHUT_China--_Fig._7_Raw_OD600_Curve_.jpg" width="724" height="484" alt=""/></div><br><div align="center">
 +
  <div align="center">Fig. 7 Raw OD600 Curve</div>
 +
</div>
 +
    <h4>5.We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures</h4>  
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f1/T--AHUT_China--_Fig._8_CFU_Result.jpg" width="724" height="420" alt=""/></div><br>
 +
    <div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e5/T--AHUT_China--_Fig._8_CFU_Result1.jpg" width="732" height="492" alt=""/></div><br>
 +
    <div align="center">
 +
  <div align="center" >Fig. 8 CFU Result</div>
 +
  <p >&nbsp;</p>
 +
<div align="center"><h2 class="title_color">Discussion</h2></div>
 +
                <p>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>            
 +
<div align="center"><h2 class="title_color">Conclusion</h2></div>
 +
                <p>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.
 +
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 +
               
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Revision as of 02:03, 5 October 2018

Royal Hotel Royal Hotel

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     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

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.