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

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                 <div align="center"> <h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ATTRIBUTION</h2></div>
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                 <div align="center"> <h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Introduction</h2></div>
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                  <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>
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Our iGEM team is from Anhui University of Technology, Ma 'anshan, China.  
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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Materials</h2></div>
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The name of our project this year is Carbon dioxide purifier. The brief introduction of the project is as follows:
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                  <p>Plate reader: Synergy H1 (Biotek)<br>
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<br/><br/>
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Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)<br>
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With greenhouse effect becoming a widespread concern in recent years, how to effectively capture CO2 has become a worldwide problem. At present, CO2 capture mostly includes absorption, adsorption and membrane methods, etc., which have problems with high cost, high energy consumption for regeneration and secondary pollution. CO2 capture using carbonic anhydrase has attracted extensive attention due to its high catalytic efficiency and environmentally friendly properties. First, our project successfully expressed wide type carbonic anhydrase in E. coli, however, its industrial application was limited due to poor stability and easy inactivation. Therefore, based on this, molecular simulation technology was used to investigate effect of amino acid residues mutation on the conformation and activity of enzyme, and the mutant carbonic anhydrase with higher thermal stability was obtained. The experimental results showed that the purified mutant carbonic anhydrase exhibited higher stability and activity than wild type carbonic anhydrase, achieving efficient capture of CO2.<br/>
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Cell culture shaker: ZWYR-200D<br><br>
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Devices:<br>
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<big><b>Overview</b></big><br/>
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Negative control ：BBa_R0040 <br>
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We were divided into groups of students in different majors, including those responsible for experiment, art design, web programming, social practice and cooperation, and mathematical modeling. <br/>
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Positive control ：BBa_I20270 <br>  
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At the same time, as we are from a country where the mother tongue is not English, we have arranged some students to be responsible for English translation and English presentation. <br/>
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Device 1： BBa_J364000  <br>
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<big><b>Experiment group</b></big><br/>
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Device 2： BBa_J364001  <br>
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The students in charge of the experiment are Pan Luyao, Liu Zhihao, Bian Weixin, Wang Xiangxuan, Hu Zhiyuan, Qin Zichen, Wang Huichao,Chen Yuru,Chen Jinrui. <br/>
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Device 3： BBa_J364002  <br>
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They completed the experimental portion of the project, building Biobricks that met the standards of iGEM. <br/>
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Device 4： BBa_J364007  <br>
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<big><b>Art design group</b></big><br/>
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Device 5： BBa_J364008  <br>
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The students responsible for art design are Sun Yu, Wang Yalong and Jiang Yu. They completed the design of poster, team flag, team banner, and team uniform, and they participated in the production of some pictures of wiki. <br/>
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Device 6： BBa_J364009  <br>
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<big><b>Wiki group</b></big><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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The students responsible for web programming students are Cao Jinhui, Zhu Dazhu, Tao Wei.<br/>
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Calibration material: Provided in the 2018 IGEM distribution kit <br>
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They did all the wiki job.<br/>  
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Microorganism: Escherichia coli DH5⍺ strains<br>
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<big><b>Modeling group</b></big><br/>
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</p><br>
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The students responsible for mathematical modeling are Wang Qiong, Zhao Lei and Ma Xiaoguang.<br/>
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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Methods</h2></div>
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They completed the mathematical modeling work related to the project, and Zhao Lei developed the gas detection device used in the cooperation activity with an aerial model team of our university. <br/>
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                  <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>
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<big><b>English group</b></big><br/>
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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Results</h2></div>
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The students responsible for English translation and presentation are Xia Yongli, Ge Yan, Chen Chen, Zhou Wan, Zhao Boya.<br/>
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                  <h4>1.OD 600 reference point</h4><p>
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They have completed all the documents on the wiki that need to be translated into English, and Xia Yongli, Ge Yan, Chen Chen, Zhou Wan and Wang Huichao will have live presentations during the competition.<br/>  
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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
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<big><b>Human practice group</b></big><br/>
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</p><br>
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The students responsible for social practice and cooperation are Fan Zixuan, Zhou Yu, Lou Feiyue.All activities of HP and collaboration are organized and conducted by them. They are also responsible for communication with iGEM teams of other colleges. <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="317" height="234" alt=""/></div><br><div align="center">Fig. 1 LUDOX correct value
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  </div>
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<big><b>Acknowledgement</b></big><br/>
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  <h4>2.Particle standard curve</h4>
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Our team consists of 8 instructors, including Xu Xiangrong,Xu Hao,Ma Liang,Liu Zi,Ruilan Cao,She Huili,Xu Xia,Xu Jian. Our PI is Xu Xiangrong and Secondary PI is Xu Hao. <br/>
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                  <p>
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We are so grateful to the instructors and advisors for their advice on our work. They provided us with laboratories and professional guidance.<br/>  
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We obtained the two Particle Standard Curve (normal and log scale).
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Prof. Xu Xiangrong provided some information of Human practice. <br/>
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</p><br>
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Prof.Xu Xia,Prof.Xu Jian,Dr.Ma Liang and Dr.Liu Zi are responsible for the guidance of the wet lab. <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="701" height="440" alt=""/></div><br><div align="center">Fig. 2 Particle Standard Curve
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Our experiment mainly carried out in Dr.Ma Liang's laboratory. At the same time, we carried out experiments with the help of some<br/> instruments in Biochemical Engineering Research Center, BERC-AHUT by Prof.Xu Xia and Prof.Xu Jian.<br/>  
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  </div>
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Prof.Cao Ruilan gave us some suggestions on English translation and English presentation <br/>
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<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">
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Dr.She Huili gave us some advice on art design. <br/>
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  Fig. 3 Particle Standard Curve (log scale)
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Dr. Xu Hao guides us the programming, mathematical modeling and Dry lab <br/>
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</div>
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Meanwhile, we invited Leng Huji, Ding Yuqing, Zou Shoushuo and Long Lijuan as advisors. They provided us some suggestions for our project.
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<h4>3.Fluorescein standard curve</h4><p>
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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>
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We obtained the two Fluorescein Standard Curve (normal and log scale).
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</div>
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</p><br>
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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="701" height="440" alt=""/></div><br><div align="center">
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  Fig. 4 Fluorescein Standard Curve
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</div>
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<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">
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  <div align="center" >Fig. 5 Fluorescein Standard Curve (log scale) </div>
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</div>
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  <h4>4.Cell measurements</h4>
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  </ol>
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<p>&nbsp;</p><br>
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<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">
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  <div align="center">Fig. 6 Fluorescence Measurements Curve</div>
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</div>
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    <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.
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</p><br>
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<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">
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  <div align="center">Fig. 7 Raw OD600 Curve</div>
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</div>
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    <h4>5.We obtained the Colony Forming Units per 0.1 OD600 E. coli cultures</h4>  
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<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>
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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="732" height="492" alt=""/></div><br>
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    <div align="center">
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  <div align="center" >Fig. 8 CFU Result</div>
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  <p >&nbsp;</p>
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<div align="center"><h2 class="title_color">Discussion</h2></div>
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                <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.  
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  </p>           
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<div align="center"><h2 class="title_color">Conclusion</h2></div>
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                <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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</p>
 
                  
 
                  
 
                  
 
                  
 
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Revision as of 03:45, 9 October 2018

Royal Hotel Royal Hotel

     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.