Difference between revisions of "Team:AHUT China/InterLab"
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<div class="section_title "> | <div class="section_title "> | ||
<div align="center"> <h2 class="title_color"> Introduction</h2></div> | <div align="center"> <h2 class="title_color"> Introduction</h2></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: 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> | |
<div align="center"><h2 class="title_color"> Materials</h2></div> | <div align="center"><h2 class="title_color"> Materials</h2></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: 18px;" > Plate reader: Synergy H1 (Biotek)<br> | |
Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)<br> | Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)<br> | ||
| − | Cell culture shaker: ZWYR-200D<br><br> | + | Cell culture shaker: ZWYR-200D<br><br></p> |
| − | Devices:<br> | + | <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 ：BBa_R0040 <br> | + | Negative control ：BBa_R0040 <br> |
| − | Positive control ：BBa_I20270 <br> | + | Positive control ：BBa_I20270 <br> |
| − | Device 1： BBa_J364000 <br> | + | Device 1： BBa_J364000 <br> |
| − | Device 2： BBa_J364001 <br> | + | Device 2： BBa_J364001 <br> |
| − | Device 3： BBa_J364002 <br> | + | Device 3： BBa_J364002 <br> |
| − | Device 4： BBa_J364007 <br> | + | Device 4： BBa_J364007 <br> |
| − | Device 5： BBa_J364008 <br> | + | Device 5： BBa_J364008 <br> |
| − | Device 6： BBa_J364009 <br> | + | Device 6： BBa_J364009 <br></p> |
| − | 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> | + | <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> | ||
Microorganism: Escherichia coli DH5⍺ strains<br> | Microorganism: Escherichia coli DH5⍺ strains<br> | ||
</p><br> | </p><br> | ||
<div align="center"><h2 class="title_color"> Methods</h2></div> | <div align="center"><h2 class="title_color"> Methods</h2></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: 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> | |
<div align="center"><h2 class="title_color"> Results</h2></div> | <div align="center"><h2 class="title_color"> Results</h2></div> | ||
| − | < | + | <h2> 1.OD 600 reference point</h2> <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;" > |
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 | 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> | </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 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> | </div> | ||
| − | < | + | <h2> 2.Particle standard curve</h2> |
| − | + | <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 obtained the two Particle Standard Curve (normal and log scale). | We obtained the two Particle Standard Curve (normal and log scale). | ||
</p><br> | </p><br> | ||
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Fig. 3 Particle Standard Curve (log scale) | Fig. 3 Particle Standard Curve (log scale) | ||
</div> | </div> | ||
| − | < | + | <h2> 3.Fluorescein standard curve</h2> <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;" > |
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> | 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). | We obtained the two Fluorescein Standard Curve (normal and log scale). | ||
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</div> | </div> | ||
| − | < | + | <h2> 4.Cell measurements</h2> |
</ol> | </ol> | ||
<p> </p><br> | <p> </p><br> | ||
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<div align="center">Fig. 6 Fluorescence Measurements Curve</div> | <div align="center">Fig. 6 Fluorescence Measurements Curve</div> | ||
</div> | </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: 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. | |
</p><br> | </p><br> | ||
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<div align="center">Fig. 7 Raw OD600 Curve</div> | <div align="center">Fig. 7 Raw OD600 Curve</div> | ||
</div> | </div> | ||
| − | < | + | <h2> 5.We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures</h2> |
<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/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"><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> | ||
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<p > </p> | <p > </p> | ||
<div align="center"><h2 class="title_color">Discussion</h2></div> | <div align="center"><h2 class="title_color">Discussion</h2></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: 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> | </p> | ||
<div align="center"><h2 class="title_color">Conclusion</h2></div> | <div align="center"><h2 class="title_color">Conclusion</h2></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: 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> | </p> | ||
Revision as of 10:26, 9 October 2018
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.