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

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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>
                   <p>We took part in the Fifth International InterLab Measurement Study which aims 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 of 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;">&nbsp;&nbsp;&nbsp;&nbsp;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 solvent absorption, physical adsorption and membrane separation, etc.The above CO2 capture technologies all have the disadvantages of high cost, low efficiency and poor circulability. These unavoidable disadvantages hinder their application in production and life. Therefore, new technologies are urgently needed, and the technology of carbonic anhydrase (CA) capture makes up for the shortage of other methods. </p>
<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;First, our project successfully expressed wild 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 project includes the following aspects: </p>
                  <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;1) molecular simulation; </p>
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;2) construction of escherichia coli strains expressing wild type and mutant CA2; </p>
Cell culture shaker: ZWYR-200D<br><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) expression and purification of CA2; </p>
Devices:<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;4) practical application of CA2: CO2 capture. </p>
Negative control :BBa_R0040 <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;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.</p>
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 device 2 shows 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 03:11, 16 October 2018

Royal Hotel Royal Hotel







     Project Overview


    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 solvent absorption, physical adsorption and membrane separation, etc.The above CO2 capture technologies all have the disadvantages of high cost, low efficiency and poor circulability. These unavoidable disadvantages hinder their application in production and life. Therefore, new technologies are urgently needed, and the technology of carbonic anhydrase (CA) capture makes up for the shortage of other methods.

    First, our project successfully expressed wild 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 project includes the following aspects:

      1) molecular simulation;

      2) construction of escherichia coli strains expressing wild type and mutant CA2;

      3) expression and purification of CA2;

      4) practical application of CA2: CO2 capture.

    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.