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| − | {{NUDT_CHINA/navbar}} | + | {{NUDT_CHINA/content}} |
| | <html> | | <html> |
| − | <style>
| + | <div class="col-md-12 column" style="width:100%;height:250px;background-size:contain;0px -100px; background-image:url(https://static.igem.org/mediawiki/2018/2/2a/T--NUDT_CHINA--banner_project_notebook.jpg);background-repeat:no-repeat;"> |
| − | #content {
| + | <p style="font-size:36px;margin: 0 0%;padding: 5% 0 0 10%;color: white;">Designed Protein Degradation Method Based on</p> |
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| + | <p style="font-size:36px;margin: 0 0%;padding: 0 0 0 10%;color: white;">Trim21 And Nanobody -- Notebook</p> |
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| − | /* Sections Header
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| − | <div class="col-md-12 column" alt="cover_photo" style="width:100%;height:250px;background-size:cover;background-position:0px -350px; background-image:url(https://static.igem.org/mediawiki/2016/thumb/3/3f/NUDT_CHINA2016_FILES-img-banner-3.0.png/800px-NUDT_CHINA2016_FILES-img-banner-3.0.png);background-repeat:no-repeat;"> | + | |
| − | <p style="font-size:36px;margin: 0 0%;padding: 8% 0 0 10%;color: white;">Development of A Novel</p> | + | |
| − | <p style="font-size:36px;margin: 0 0%;padding: 0 0 0 10%;color: white;">Blood-MicroRNA Handy Detection System with CRISPR</p> | + | |
| | </div> | | </div> |
| − |
| |
| | <div class="col-md-push-1 col-md-10"> | | <div class="col-md-push-1 col-md-10"> |
| − | <h1 style="font-size: 40px;font-weight: 900"></h1> | + | <h1 style="font-size: 40px;font-weight: 1200"></h1> |
| | <p alt="content_add" style="font-size: 20px;"> | | <p alt="content_add" style="font-size: 20px;"> |
| − | Chuanyang Liu, Changsong Gao, Chushu Zhu and Qijie Xu completed most of the experiments. | + | Jiaxin Ma ,Tianyi Zhang,Chenyu Lu and Chenxing Sun completed most of the experiments. |
| | </p> | | </p> |
| | | | |
| | </div> | | </div> |
| | | | |
| − | | + | <div class="col-md-push-1 col-md-10"> |
| − | | + | |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">Chuanyang Liu </h2> | + | |
| | <center> | | <center> |
| − | <table> | + | <table class="table table-striped"> |
| | + | <caption style="font-size: 25px">Jiaxin Ma</caption> |
| | <tbody> | | <tbody> |
| | <tr> | | <tr> |
| Line 135: |
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| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>6.15-7.4</strong></p> | + | <p><strong>6.15-7.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the culture of A549 cell and optimization of culture condition with Changsong Gao</p> | + | <p>Construct plasmid expressing GFPnano-IgG1Fc and Trim21 with Tianyi Zhang.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>7.5-</strong><strong>8.13</strong></p> | + | <p><strong>7.4-8.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Work on Western Blot assay for target gene expression with Changsong Gao</p> | + | <p>Work on the optimization of transfection and fluorescence-detecting conditions in Hela and A549 cells with Tianyi Zhang.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.</strong><strong>14-8.26</strong></p> | + | <p><strong>8.4-8.15</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Do Western Blot assay EMT markers with Changsong Gao</p> | + | <p>Work on Western Blot assay for target gene (GFP) expression and fluorescence detection with Tianyi Zhang.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.27-9.22</strong></p> | + | <p><strong>8.16-8.28</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Cell proliferation and migration assay of miR-214 Locker group with Changsong Gao</p> | + | <p>Work on repeated validation of GFP interfering efficiency with Tianyi Zhang.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>9.22-10.14</strong></p> | + | <p><strong>8.29-9.22</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Cell proliferation and migration assay of miR-654 Locker group with Changsong Gao</p> | + | <p>Work on transfection optimization, EMT-related Markers detection of ErbB3-interfered cells with Tianyi Zhang.</p> |
| | + | </td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td width="153"> |
| | + | <p><strong>9.22-10.10</strong></p> |
| | + | </td> |
| | + | <td width="538"> |
| | + | <p>Work on the proliferation and migration assay of cells with ErbB3 interfered with Tianyi Zhang.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | </tbody> | | </tbody> |
| | </table> | | </table> |
| − | </center> | + | </center> |
| − | <h2 class="content-subsub">Changsong Gao</h2> | + | </div> |
| | + | <div class="col-md-push-1 col-md-10"> |
| | <center> | | <center> |
| − | <table> | + | <table class="table table-striped"> |
| | + | <caption style="font-size: 25px">Tianyi Zhang</caption> |
| | <tbody> | | <tbody> |
| | <tr> | | <tr> |
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| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>6.15-7.4</strong></p> | + | <p><strong>6.15-7.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the culture of A549 cell and optimization of culture condition with Chuanyang Liu</p> | + | <p>Construct plasmid expressing GFPnano-IgG1Fc and Trim21 with Jiaxin Ma. </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>7.5-</strong><strong>8.13</strong></p> | + | <p><strong>7.4-8.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Work on Western Blot assay for target gene expression with Chuanyang Liu</p> | + | <p>Work on the optimization of transfection and fluorescence-detecting conditions in Hela and A549 cells with Jiaxin Ma.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.</strong><strong>14-8.26</strong></p> | + | <p><strong>8.4-8.15</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Do Western Blot assay EMT markers with Chuanyang Liu</p> | + | <p>Work on Western Blot assay for target gene (GFP) expression and fluorescence detection with Jiaxin Ma.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.27-9.22</strong></p> | + | <p><strong>8.16-8.28</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Cell proliferation and migration assay of miR-214 Locker group with Chuanyang Liu</p> | + | <p>Work on repeated validation of GFP interfering efficiency with Jiaxin Ma.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>9.22-10.14</strong></p> | + | <p><strong>8.29-9.22</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Cell proliferation and migration assay of miR-654 Locker group with Chuanyang Liu</p> | + | <p>Work on transfection optimization, EMT-related Markers detection of ErbB3-interfered cells with Jiaxin Ma.</p> |
| | + | </td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td width="153"> |
| | + | <p><strong>9.22-10.10</strong></p> |
| | + | </td> |
| | + | <td width="538"> |
| | + | <p>Work on the proliferation and migration assay of cells with ErbB3 interfered with Jiaxin Ma.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | </tbody> | | </tbody> |
| | </table> | | </table> |
| − | </center> | + | </center> |
| − | <h2 class="content-subsub">Chushu Zhu</h2> | + | </div> |
| | + | <div class="col-md-push-1 col-md-10"> |
| | <center> | | <center> |
| − | <table> | + | <table class="table table-striped"> |
| | + | <caption style="font-size: 25px">Chenyu Lu</caption> |
| | <tbody> | | <tbody> |
| | <tr> | | <tr> |
| Line 245: |
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| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>6.15-7.4</strong></p> | + | <p><strong>6.15-7.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Phosphorylation and annealing of the oligos with Qijie Xu</p> | + | <p>Validation of plasmid expression GFP-nano-IgGFc and Trim21, culture of Hela cells with Chenxing Sun.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>7.5-</strong><strong>8.13</strong></p> | + | <p><strong>7.4-8.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Work on miRNA locker assembly with Qijie Xu</p> | + | <p>Hela Cell culture and preparatory works on fluorescence detection with Chenxing Sun. </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.</strong><strong>14-8.26</strong></p> | + | <p><strong>8.4-8.28</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Asymmetric PCR to generate ssDNAs from dsDNAs with Qijie Xu</p> | + | <p>Construct plasmid expression ErbB3-IgG1Fc and trim21, and construct Parts with Chenxing Sun.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.27-9.22</strong></p> | + | <p><strong>8.29-9.22</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Construct parts with Qijie Xu</p> | + | <p>Construct Parts with Chenxing Sun.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>9.22-10.14</strong></p> | + | <p><strong>9.22-10.10</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Work on the experimental validation of parts with Qijie Xu</p> | + | <p>Work on the experimental validation of parts with Chenxing Sun.</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | </tbody> | | </tbody> |
| | </table> | | </table> |
| − | </center> | + | </center> |
| − | <h2 class="content-subsub">Qijie Xu</h2> | + | </div> |
| | + | |
| | + | <div class="col-md-push-1 col-md-10"> |
| | <center> | | <center> |
| − | <table> | + | <table class="table table-striped"> |
| | + | <caption style="font-size: 25px">Chenxing Sun</caption> |
| | <tbody> | | <tbody> |
| | <tr> | | <tr> |
| Line 300: |
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| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>6.15-7.4</strong></p> | + | <p><strong>6.15-7.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Phosphorylation and annealing of the oligos with Chushu Zhu</p> | + | <p>Validation of plasmid expression GFP-nano-IgGFc and Trim21, culture of Hela cells with Chenyu Lu . </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>7.5-</strong><strong>8.13</strong></p> | + | <p><strong>7.4-8.3</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Work on miRNA locker assembly with Chushu Zhu</p> | + | <p>Hela Cell culture and preparatory works on fluorescence detection with Chenyu Lu . </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.</strong><strong>14-8.26</strong></p> | + | <p><strong>8.4-8.28</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Finish the Asymmetric PCR to generate ssDNAs from dsDNAs with Chushu Zhu</p> | + | <p>Construct plasmid expression ErbB3-IgG1Fc and trim21, and construct Parts with Chenyu Lu .</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>8.27-9.22</strong></p> | + | <p><strong>8.29-9.22</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Construct parts with Chushu Zhu</p> | + | <p>Construct Parts with Chenyu Lu .</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td width="153"> | | <td width="153"> |
| − | <p><strong>9.22-10.14</strong></p> | + | <p><strong>9.22-10.10</strong></p> |
| | </td> | | </td> |
| | <td width="538"> | | <td width="538"> |
| − | <p>Work on the experimental validation of parts with Chushu Zhu</p> | + | <p>Work on the experimental validation of parts with Chenyu Lu .</p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | </tbody> | | </tbody> |
| − | </table></center>
| |
| − | <div class="col-md-push-1 col-md-10">
| |
| − | <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Methods and Design</h2>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">1. InterLab Parts</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">Positive Control (BBa_I20270) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">NegativeControl (BBa_R0040) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">Test Device1 (BBa_J364000) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">Test Device2 (BBa_J364001) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">Test Device3 (BBa_J364002) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">Test Device4 (BBa_J364007) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">Test Device5 (BBa_J364008) </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">Test Device6 (BBa_J364009) </p>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2. Preparation</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">To start with, our team transformed E.coli strain DH5α with the provided plasmids, namely Test Device 1,2,3,4,5,6, Positive Control, and Negative Control. As long as colonies had emerged on Cm<sup>+</sup> Resistance Media, we picked up 2 colonies in each petrie dish and cultured them at 37℃ with 220 rpm frequency for 14 hours. When the bacteria solutions were turbid enough, we began the following process.
| |
| − | </p>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">3. OD600 Reference Point</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">With plate reader, we measured Abs600 of the LUDOX and H2O. The H2O measurement served as the background. Both included 4 technical replicates to enhance the reliability of the results. Comparing to the standard OD600 reference given, which was 0.0425, we were able to achieve a ratio between OD600 and Abs600. The ratio was essential in converting Abs600 raw measurements into standard OD600 records.
| |
| − | </p>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">4. Fluorescein Standard Curve</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">Different concentrations of fluorescein were obtained by 2-fold serial dilution. In the first pipet, 200μL Fluorescein 1×stock solution was added. By removing half of the 200μL solution in previous pipets to later ones which already contained 100μL PBS, we were able to generate 11 solutions in half-descending fluorescence concentration. We also included another pipet with 100μL PBS only as blank. Within each concentration, we performed 4 replicates to calculate the more representative mean. After the results were recorded, all fluorescein concentrations were divided by average fluorescence measurement, of which the medium-high mean were calculated to diminish operation errors. This mean point would be used to set up the conversion between fluorescein concentration and fluorescence measurements in later steps.
| |
| − | </p>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">5. OD600 and Fluorescence Measurements</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">We tracked the original OD600 of the 16 samples so as to dilute them into 0.02. Once the original values were available, we integrated them into the dilution calculation sheet and followed the suggested volume to dilute each sample. After dilution, we confirmed that OD600 had reached exactly 0.02 or around. Our team set that time as T=0(h) and measured average OD600 from 4 replicates of each 16 samples to reduce technical error. Other 4 replicates were used for T=0(h) fluorescence measurements, with the same equipment and settings in Step 3. Henceforward, we recorded the OD600 and fluorescence when T=0 and T=6.
| |
| − | </p>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">6. Equipments and Settings</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">To obtain OD600 measurement, we employed Multiscan FC. Single wavelength of 600nm was used, and path length correction was turned off. To reduce measurement error, we also added a dynamic circulation of 5 times, with 2-second intervals.
| |
| − | </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">The fluorescence measurement was obtained through Fluoroskan Ascent FL and Thermo Fisher. The filters used were 485nm and 538nm for excitation and emission respectively. The measurement cycled 5 times with intervals of 2 second.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-push-1 col-md-10">
| |
| − | <h2 style="font-size: 32px;margin-bottom: 10px;margin-top: 15px;">Results & Discussion</h2>
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| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">Calibration</h3>
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| − | <p alt="content_add" style="font-size: 18px;">Calibration1: OD600 Reference point – LUDOX Protocol </p>
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| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | We used LUDOX CL-X (45% colloidal silica suspension) , ddH2O and 96 well plate(black with clear flat bottom preferred) to get the conversion factor, which can transform Abs600 measurements into comparable OD600 measurements.
| |
| − | </p>
| |
| − | <table class="table table-striped">
| |
| − | <caption>Fig.1 OD600 reference point</caption>
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th></th>
| |
| − | <th>LUDOX CL-X</th>
| |
| − | <th>H2O</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td>Replicate 1</td>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.062</p>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.039</p>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.060</p>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.039</p>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.066</p>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.038</p>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.061</p>
| |
| − | <td bgcolor="#00ccff" >
| |
| − | <p>0.038</p>
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| − | </tr>
| |
| − | <tr>
| |
| − | <td>Arith. Mean</td>
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| − | <td bgcolor="#ffcc00">
| |
| − | <p>0.062</p>
| |
| − | <td bgcolor="#ffcc00">
| |
| − | <p>0.039</p>
| |
| − | </tr>
| |
| − | <tr>
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| − | <td>Corrected Abs600</td>
| |
| − | <td bgcolor="#ffcc00">
| |
| − | <p>0.024</p>
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| − | <td></td>
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| − | </tr>
| |
| − | <tr>
| |
| − | <td>Reference OD600</td>
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| − | <td bgcolor="#ffcc00">
| |
| − | <p>0.063</p>
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| − | <td></td>
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| − | </tr>
| |
| − | <tr>
| |
| − | <td>OD600/Abs600</td>
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| − | <td bgcolor="#ffcc00">
| |
| − | <p>2.654</p>
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| − | <td></td>
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| − | </tr>
| |
| − | </tbody>
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| − | </table>
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| − | </div>
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| − |
| |
| − | <div class="col-md-push-1 col-md-10">
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| − | <p alt="content_add" style="font-size: 18px;">Calibration2: Particle Standard Curve – Microsphere Protocol </p>
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| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | Following the protocol, we prepared a dilution series of monodisperse silica microspheres and measure the Abs600 in our plate reader. We got a particle standard curve and transferred it into log scale, which are shown as below.
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="col-md-12 column" alt = "main_content">
| |
| − | <div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
| |
| − | <img alt="300x200" src="https://static.igem.org/mediawiki/2018/thumb/3/38/T--NUDT_CHINA--Particle.png/800px-T--NUDT_CHINA--Particle.png" />
| |
| − | <p alt="photo_detail" style="text-align: center;">Fig.2 Particle standard curve</p>
| |
| − | </div>
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| − |
| |
| − |
| |
| − | <div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
| |
| − | <img alt="800x433" src="https://static.igem.org/mediawiki/2018/5/5f/T--NUDT_CHINA--Particle_standard_curve_%28log_scale%29.png" />
| |
| − | <p alt="photo_detail" style="text-align: center;">Fig.3 Particle standard curve (log scale)</p>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-push-1 col-md-10">
| |
| − | <p alt="content_add" style="font-size: 18px;">Calibration3: Fluorescence standard curve – Fluorescein Protocol </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | We did a dilution series of fluorescein in four replicates. After recording the measurements, we generated a standard curve of fluorescence for fluorescein concentration and its log scale version, which are shown as below.
| |
| − | </p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
| |
| − | <img alt="300x200" src="https://static.igem.org/mediawiki/2018/6/68/T--NUDT_CHINA--Particle_standard_curve.png" />
| |
| − | <p alt="photo_detail" style="text-align: center;">Fig.4 Particle standard curve</p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-8 col-md-push-2 thumbnail" alt="content_pictute">
| |
| − | <img alt="300x200" src="https://static.igem.org/mediawiki/2018/4/4a/T--NUDT_CHINA--Flourescein_standard_curve_%28log_scale%29.png" />
| |
| − | <p alt="photo_detail" style="text-align: center;">Fig.5 Particle standard curve (log scale)</p>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-md-push-1 col-md-10">
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">Cell measurement</h3>
| |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2.1 Abs600</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | We have read two plates at the time point: 0 and 6 hours with the same conditions in our calibration measurements.
| |
| − | </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | We measured both fluorescence and absorbance of 2 biological replicates and 4 technical replicates. Abs600 values for device 5 are almost the lowest while the values of device 3 are almost the highest.
| |
| − | </p>
| |
| − | <table class="table table-striped">
| |
| − | <caption>Abs600 Raw Readings:</caption>
| |
| − | <thead>
| |
| − | <tr>
| |
| − | <th> </th>
| |
| − | <th> </th>
| |
| − | <th>Hour 0</th>
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| − | <th>Hour 6</th>
| |
| − | </tr>
| |
| − | </thead>
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Neg. Control</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.068</td>
| |
| − | <td>0.297</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.069</td>
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| − | <td>0.285</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.068</td>
| |
| − | <td>0.291</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.072</td>
| |
| − | <td>0.286</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Pos. Control</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.072</td>
| |
| − | <td>0.296</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.071</td>
| |
| − | <td>0.288</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.075</td>
| |
| − | <td>0.284</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.068</td>
| |
| − | <td>0.285</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device1</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.068</td>
| |
| − | <td>0.239</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.22</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.073</td>
| |
| − | <td>0.223</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.069</td>
| |
| − | <td>0.224</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device2</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.066</td>
| |
| − | <td>0.268</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.071</td>
| |
| − | <td>0.27</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.068</td>
| |
| − | <td>0.268</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.066</td>
| |
| − | <td>0.273</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device3</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.069</td>
| |
| − | <td>0.425</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.358</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.072</td>
| |
| − | <td>0.343</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.074</td>
| |
| − | <td>0.347</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device4</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.21</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.217</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.217</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.07</td>
| |
| − | <td>0.221</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device5</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.066</td>
| |
| − | <td>0.161</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.155</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.069</td>
| |
| − | <td>0.16</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.066</td>
| |
| − | <td>0.164</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> Device6</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.294</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.068</td>
| |
| − | <td>0.284</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.067</td>
| |
| − | <td>0.286</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.071</td>
| |
| − | <td>0.285</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <th rowspan="4" style="vertical-align: inherit;font-size: 120%;"> LB+Chior(blank)</th>
| |
| − | <td> Replicate 1</td>
| |
| − | <td>0.048</td>
| |
| − | <td>0.044</td>
| |
| − | </tr>
| |
| − | <td>Replicate 2</td>
| |
| − | <td>0.048</td>
| |
| − | <td>0.046</td>
| |
| − | </tr>
| |
| − | <td>Replicate 3</td>
| |
| − | <td>0.047</td>
| |
| − | <td>0.046</td>
| |
| − | </tr>
| |
| − | <td>Replicate 4</td>
| |
| − | <td>0.049</td>
| |
| − | <td>0.046</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| | </table> | | </table> |
| − | <p alt="content_add" style="font-size: 18px;">
| + | </center> |
| − | We also used colony 2 series to do the same experiments. The features are similar to colony group. However, Abs600 value of device 2 reach the same level of device 3.
| + | </div> |
| − | </p>
| + | |
| − | <h3 style="font-size: 28px;margin-bottom: 10px;margin-top: 15px;">2.2 Fluorescence</h3>
| + | |
| − | <p alt="content_add" style="font-size: 18px;">
| + | |
| − | We used same colonies to measure their fluorescence values of device.
| + | |
| − | </p>
| + | |
| − | <p alt="content_add" style="font-size: 18px;">
| + | |
| − | As it is shown in figure 8, Fluorescence value of device 1 is the highest while the value of device 3 is the lowest, which approaches to the value of counterpart of negative control.
| + | |
| − | </p>
| + | |
| − | <p alt="content_add" style="font-size: 18px;">
| + | |
| − | At the second time, results of almost devices are in agreement to the first time, nevertheless, fluorescence value for device 2, which going to the highest.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | | | |
| − | <div class="col-mdh3 style="font-siz-push-1 col-md-10">
| |
| − | <e: 28px;margin-bottom: 10px;margin-top: 15px;">Protocol:</h3>
| |
| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | We measured the Abs600 of our cell cultures and then used the appropriate ratio between Abs600 and OD600 to calculate the OD600 value. After getting the results, we diluted our overnight culture to OD600=1.0 for each culture and checked it.
| |
| − | </p>
| |
| − | <p alt="content_add" style="font-size: 18px;">
| |
| − | Finally, we counted the colonies on each plate with fewer than 300 colonies. Then, we multiplied by the Final Dilution Factor: 8 . We recorded the CFU per 1mL of each OD600 = 0.1 culture.
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
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