Difference between revisions of "Team:AHUT China/New Parts"

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            <div align="center" class="section4"><img src="https://static.igem.org/mediawiki/2018/c/c1/T--AHUT_China--_chilun.jpg" width="332" height="452" alt=""/></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 style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-style: normal; font-weight: 400; font-size: 36px; text-align: center;"> &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> 8.29</strong></h2>
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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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                <hr>
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<div align="center"><h2 class="title_color">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Materials</h2></div>
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                <nbsp><nbsp>
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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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Pick</span> Clon  </span></p>
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Plate reader plates: Corning 3603 96-Well Microplates (black plates with clear flat bottom)<br>
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<br><br>
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Cell culture shaker: ZWYR-200D<br><br>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/0/02/T--AHUT_China--829.png" width="450" height="77" alt=""/></div><br>
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Devices:<br>
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    </div>
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Negative control ：BBa_R0040 <br>
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<div class="container">
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Positive control ：BBa_I20270 <br>  
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              <div class="section_title ">
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Device 1： BBa_J364000  <br>
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                  <p style="text-align: center; font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 36px;"><strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400; color: #000000;">8.30</strong></p>
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Device 2： BBa_J364001  <br>
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                </h2>
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Device 3： BBa_J364002  <br>
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                <hr>
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Device 4： BBa_J364007  <br>
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Device 5： BBa_J364008  <br>
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                  <nbsp><nbsp>
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Device 6： BBa_J364009  <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: 16px; ">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">Pilot </span> Expression of Protein<br>&nbsp;&nbsp;&nbsp;“IPTG+ 4h”“IPTG- 4h”</p>  
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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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<p>&nbsp;</p> <p>&nbsp;</p>            
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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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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/4/44/T--AHUT_China--830.png" width="420" height="77" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;After the rocker culture is 4 H, add IPTG(final concentration is 0.5 mm) and then the rocker culture is 4 hours. Each of the two tubes absorbs 200 μL of bacterial fluid, and in the frozen centrifuge, it is 4 °C and 12000 rpm centrifuge. 10 minutes, go to the clear, It is resuspended with 100 μL 1x SDS Loading buffer, then heated in a water bath pot at 100 °C for 10 minutes and stored in a -20 °C refrigerator.</p>  
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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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</p>
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            </div>
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           </div>
 
           </div>
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                        <div class="container">
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        </section>
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              <div class="section_title ">
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> 8.31</strong></h2>
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                <hr>
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                <nbsp><nbsp>
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Protein</span> extraction <br>&nbsp;&nbsp;&nbsp;&nbsp;CA2L203K</p> 
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<p>&nbsp;</p> <p>&nbsp;</p> 
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/2/2d/T--AHUT_China--831.11.png" width="761" height="76" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/eb/T--AHUT_China--831.12.png" width="657" height="476" alt=""/></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: 16px;">&nbsp;&nbsp;&nbsp;&nbsp;CA2WT<br>&nbsp;&nbsp;&nbsp;&nbsp;CA2L203K</p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/ee/T--AHUT_China--831.2.png" width="998" height="75" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/3/3f/T--AHUT_China--831.22.png" width="703" height="497" alt=""/></div>
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<br><br><br>
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        <div class="container">
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              <div class="section_title ">
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;"> 9.01</strong></h2>
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                <hr>
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                <nbsp><nbsp>
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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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">Purification</span>&nbsp;of Protein <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Prior to purification</p>
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<br>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/c/cc/T--AHUT_China--91.1.png" width="974" height="74" alt=""/></div>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/7/7b/T--AHUT_China--91.2.png" width="657" height="476" alt=""/></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: 14px;"> <span style="font-size: 16px">&nbsp;&nbsp;&nbsp;&nbsp;Purified</p><br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8a/T--AHUT_China--91.3.png" width="564" height="75" alt=""/></div>
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  <div align="center"><img src="https://static.igem.org/mediawiki/2018/6/65/T--AHUT_China--91.4.png" width="373" height="431" alt=""/></div><br>
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<br><br> <div class="container">
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              <div class="container">
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              <div class="section_title ">
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">9.03</strong></h2>
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                <hr>
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                <nbsp><nbsp>
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Western</span> blot identification of proteins<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CA2L203K&nbsp;&nbsp;&nbsp;&nbsp;CA2WT</p>  <br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/60/T--AHUT_China--93.1.png" width="971" height="167" alt=""/></div> <br>
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<br><br>               
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<div class="container">
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              <div class="section_title ">
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">9.04</strong></h2>
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                <hr>
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                <nbsp><nbsp>
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Quantification</span> of protein concentrations</p> 
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<br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/6/6e/T--AHUT_China--93.2.png" width="908" height="189" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/8b/T--AHUT_China--93.3.png" width="754" height="343" alt=""/></div><br>
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<br><br>
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<div class="container">
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              <div class="section_title ">
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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-style: normal; font-weight: 400; font-size: 36px; text-align: center;">  &nbsp;&nbsp;&nbsp;<strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400;color: #000000;">9.05</strong></h2>
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                <hr>
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                <nbsp><nbsp>
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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: 16px;">&nbsp;&nbsp;&nbsp;<span style="font-size: 24px">&nbsp;Colorimetric</span> enzyme activity<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 250 μl saturated CO2 solution</p> 
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<br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--AHUT_China--95.1.png" width="789" height="328" alt=""/></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/8/82/T--AHUT_China--95.2.png" width="669" height="347" alt=""/></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: 16px;">&nbsp;&nbsp;&nbsp;According to the average calculated in the table above, the unit enzyme activity of the mutated carbonic anhydride enzyme is 0.3710(10 5 WAU/μg), and the unit enzyme activity of wild carbonic anhydride enzyme is 0.1755(10 5 WAU/μg).<br>
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Test esterase activity<br>
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Determination curve without carbonated anhydrase:</p> <br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/f6/T--AHUT_China--95.3.png" width="903" height="296" alt=""/></div><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: 16px;">&nbsp;&nbsp;&nbsp;Determination curve with carbonated anhydrase（CA2WT）</p> <br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/e/e8/T--AHUT_China--95.4.png" width="929" height="330" alt=""/></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: 16px;">&nbsp;&nbsp;&nbsp;Determination curve with carbonated anhydrase（CA2L203K）</p> <br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/0/0c/T--AHUT_China--95.5.png" width="893" height="306" alt=""/></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: 16px;">&nbsp;&nbsp;&nbsp;Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min<br>
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&nbsp;&nbsp;&nbsp;The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min.
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</p> <br>
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Revision as of 05:01, 12 October 2018

Royal Hotel Royal Hotel

    8.29

     Pick Clon




8.30

    Pilot Expression of Protein
   “IPTG+ 4h”“IPTG- 4h”

 

 


   After the rocker culture is 4 H, add IPTG(final concentration is 0.5 mm) and then the rocker culture is 4 hours. Each of the two tubes absorbs 200 μL of bacterial fluid, and in the frozen centrifuge, it is 4 °C and 12000 rpm centrifuge. 10 minutes, go to the clear, It is resuspended with 100 μL 1x SDS Loading buffer, then heated in a water bath pot at 100 °C for 10 minutes and stored in a -20 °C refrigerator.

    8.31

    Protein extraction
    CA2L203K

 

 

    CA2WT
    CA2L203K




    9.01

    Purification of Protein
     Prior to purification


    Purified





   9.03

    Western blot identification of proteins
     CA2L203K    CA2WT





   9.04

    Quantification of protein concentrations





   9.05

    Colorimetric enzyme activity
     Add 250 μl saturated CO2 solution


   According to the average calculated in the table above, the unit enzyme activity of the mutated carbonic anhydride enzyme is 0.3710(10 5 WAU/μg), and the unit enzyme activity of wild carbonic anhydride enzyme is 0.1755(10 5 WAU/μg).
Test esterase activity
Determination curve without carbonated anhydrase:



   Determination curve with carbonated anhydrase（CA2WT）


   Determination curve with carbonated anhydrase（CA2L203K）


   Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min
   The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min.