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                 <div align="center"> <h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Introduction</h2></div>
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                 <div align="center"> <h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Project Overview</h2></div><hr>
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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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                <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;">There is an excessive volume of greenhouse gases in the atmospheric system, which contain about 77% CO<span style="font-size: 14px">2</span>, and broad consensus that this will have serious consequences in terms of climate change. A number of CO<span style="font-size: 14px">2</span> sequestration methods have been proposed in order to capture and concentrate CO<span style="font-size: 14px">2</span>. The biomimetic approach via the use of an enzyme, namely carbonic anhydrase, offers important advantages if compared to the other methods for CO<span style="font-size: 14px">2</span> capturing, such as safe, high catalytic efficiency, and environment friendly. In our project, we use human carbonic anhydrase 2 (CA2) to absorb CO<span style="font-size: 14px">2</span>, however, wild-type CA2 usually lose their activity between 55 and 65 °C, which limits its application for the absorption of CO<span style="font-size: 14px">2</span> under industrial operating conditions. Therefore, this project uses molecular simulation technology to design a high-efficiency and stable carbonic anhydrase by improving its catalytic properties and biological stability for CO<span style="font-size: 14px">2</span> capture, including the following aspects: </p>
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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Materials</h2></div>
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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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1) Molecular simulation; </p>
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                  <p>Plate reader: Synergy H1 (Biotek)<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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) Engineered E. coli strains expressing wild-type and mutant CA2; </p>
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  Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)<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;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3) Application of mutant CA2 for CO<span style="font-size: 14px">2</span> capture. </p>
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Cell culture shaker: ZWYR-200D<br><br>
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Devices:<br>
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Negative control ：BBa_R0040 <br>
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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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Device 6： BBa_J364009  <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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Calibration material: Provided in the 2018 IGEM distribution kit <br>
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Microorganism: Escherichia coli DH5⍺ strains<br>
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</p><br>
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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Methods</h2></div>
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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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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Results</h2></div>
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                  <h4>1.OD 600 reference point</h4><p>
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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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</p><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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  <h4>2.Particle standard curve</h4>
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                  <p>
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We obtained the two Particle Standard Curve (normal and log scale).
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</p><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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  </div>
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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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  Fig. 3 Particle Standard Curve (log scale)
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</div>
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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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</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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Latest revision as of 15:33, 17 October 2018

Royal Hotel Royal Hotel







     Project Overview

There is an excessive volume of greenhouse gases in the atmospheric system, which contain about 77% CO2, and broad consensus that this will have serious consequences in terms of climate change. A number of CO2 sequestration methods have been proposed in order to capture and concentrate CO2. The biomimetic approach via the use of an enzyme, namely carbonic anhydrase, offers important advantages if compared to the other methods for CO2 capturing, such as safe, high catalytic efficiency, and environment friendly. In our project, we use human carbonic anhydrase 2 (CA2) to absorb CO2, however, wild-type CA2 usually lose their activity between 55 and 65 °C, which limits its application for the absorption of CO2 under industrial operating conditions. Therefore, this project uses molecular simulation technology to design a high-efficiency and stable carbonic anhydrase by improving its catalytic properties and biological stability for CO2 capture, including the following aspects:

      1) Molecular simulation;

      2) Engineered E. coli strains expressing wild-type and mutant CA2;

      3) Application of mutant CA2 for CO2 capture.