Difference between revisions of "Team:Tianjin/Measurement"

 
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                     <p>Measurment</p>
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                     <p>MEASUREMENT</p>
 
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                     In pursuit of a rigorously scientific result, we have adopted multiple measuring approaches throughout the whole project, among which two predominant methods described in extreme detail in wiki, would inspire relative researchers.<br><br>
 
                     In pursuit of a rigorously scientific result, we have adopted multiple measuring approaches throughout the whole project, among which two predominant methods described in extreme detail in wiki, would inspire relative researchers.<br><br>
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                     As two sophisticated and renowned reporters in biotechnological domain, fluorescent protein and luciferase were opted to illustrate the effectiveness of our constructed oscillating system, detected by combined means and instrument. Mcherry and EYFP, two selected fluorescent reporters were measured by both fluorescence spectrophotometer and Leica DMi8 respectively. Leica DMi8 allows us to visually and intuitively see the expression of the fluorescent protein EYFP in Saccharomyces cerevisiae. In terms of luciferase, in addition to commonly used firefly luciferase, a courageous attempt we made was to choose a novel luciferase Nanoluc and its live cell substrate in spite of no records of the usefulness in Saccharomyces cerevisiae before and obtained expected outcome successfully.  
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                     As two sophisticated and renowned reporters in biotechnological domain, fluorescent protein and luciferase were opted to illustrate the effectiveness of our constructed oscillating system, detected by combined means and instrument. Mcherry and EYFP, two selected fluorescent reporters were measured by both fluorescence spectrophotometer and Leica DMi8 respectively. Leica DMi8 allows us to visually and intuitively see the expression of the fluorescent protein EYFP in <em>Saccharomyces cerevisiae</em> In terms of luciferase, in addition to commonly used firefly luciferase, a courageous attempt we made was to choose a novel luciferase NanoLuc and its live cell substrate in spite of no records of the usefulness in <em>Saccharomyces cerevisiae</em> before and obtained expected outcome successfully.  
 
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                                       To achieve the best measurement of our oscillating system, we chose two different kinds of reporter gene as our downstream reporter, fluorescent protein gene and luciferase gene,  which are both widely used in the biotechnological labs.
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                                       To achieve the best measurement of our oscillatory system, we chose two different kinds of reporter gene as our downstream reporters, fluorescent protein gene and luciferase gene,  which are both widely used in the biotechnological labs.
 
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                                     <p>Figure 1. Mechanism of fluorescent proteins</p>
 
                                     <p>Figure 1. Mechanism of fluorescent proteins</p>
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                                       Luciferase is a popular choice as a reporter for gene expression because functional enzyme is created immediately upon translation and the assay is rapid, reliable and easy to perform.<sup><a href="#re1">[1,2]</a></sup> The fluorescent output of luciferase gene is due to the chemical reaction between luciferase and corresponding substrate, which can be easily detected by the luminoskan microplate reader.<br>
 
                                       Luciferase is a popular choice as a reporter for gene expression because functional enzyme is created immediately upon translation and the assay is rapid, reliable and easy to perform.<sup><a href="#re1">[1,2]</a></sup> The fluorescent output of luciferase gene is due to the chemical reaction between luciferase and corresponding substrate, which can be easily detected by the luminoskan microplate reader.<br>
−
                                       We used firefly luciferase(Fluc) gene purchased from Promega and Nanoluc luciferase(Nluc)  gene submitted by Team Tuebingen 2015 as another two reporters for our KaiABC system.
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                                       We used firefly luciferase(Fluc) gene purchased from Promega and NanoLuc luciferase(Nluc)  gene submitted by Team Tuebingen 2015 as another two reporters for our KaiABC system.
 
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                                       When reconstructed in <em>Escherichia coli</em>,&nbsp;KaiABC&nbsp;oscillator showed a periodicity of 24 hours<sup><a href="#re3">[3]</a></sup>. To best verify our system can function in <em>Saccharomyces cerevisiae</em>&nbsp;, we planned to measure our system using different reporter at <strong>3-hour intervals for 1 day, 2 days, 3 days respectively</strong><strong>.</strong><br>
 
                                       When reconstructed in <em>Escherichia coli</em>,&nbsp;KaiABC&nbsp;oscillator showed a periodicity of 24 hours<sup><a href="#re3">[3]</a></sup>. To best verify our system can function in <em>Saccharomyces cerevisiae</em>&nbsp;, we planned to measure our system using different reporter at <strong>3-hour intervals for 1 day, 2 days, 3 days respectively</strong><strong>.</strong><br>
 
                                       As for the measurement of fluorescent proteins, we not only used the common detecting instrument, fourescence spectrophotometer, but also make an attempt to record the fluorescent variation via the cutting-edge Leica DMi8 inverted microscope.<br>
 
                                       As for the measurement of fluorescent proteins, we not only used the common detecting instrument, fourescence spectrophotometer, but also make an attempt to record the fluorescent variation via the cutting-edge Leica DMi8 inverted microscope.<br>
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                                       Another thing worth mentioning is that, we bought all of the luciferase assay system from company Promega, among them there was a new kind of Nanoluc&nbsp;luciferase system----Nano-Glo<sup>&reg;</sup>&nbsp;Live Cell Assay System, which allows experimenters to detect live cell luminescent signals without lytic process, suitable for the 3-day assay. Despite there is no reference indicating that this product, a commonly used assay system in mammalian cells, could be used in<em>&nbsp;</em><em>Saccharomyces cerevisiae</em>, we decided to take a try and have successfully proved its validity. Consequently, we chose Nluc solely to complete our long time luminescent detection .
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                                       Another thing worth mentioning is that, we bought all of the luciferase assay system from company Promega, among them there was a new kind of NanoLuc&nbsp;luciferase system----Nano-Glo<sup>&reg;</sup>&nbsp;Live Cell Assay System, which allows experimenters to detect live cell luminescent signals without lytic process, suitable for the 3-day assay. Despite there is no reference indicating that this product, a commonly used assay system in mammalian cells, could be used in<em>&nbsp;</em><em>Saccharomyces cerevisiae</em>, we decided to take a try and have successfully proved its validity. Consequently, we chose Nluc solely to complete our long time luminescent detection .
 
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                                         </thead>
 
                                         </thead>
 
                                         <tbody>
 
                                         <tbody>
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                                             <tr><tr><td rowspan="4" width="154"><p><strong>Experimental</strong></p><br><br><p><strong>Group</strong></p></td><td width="468"><p>pABaC +pCiRbS +gal1+mCherry</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS +gal1+mCherry</p></td></tr><tr><td width="468"><p>pABaC +pCiRbS+gal1+EYFP</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS +gal1+EYFP</p></td></tr><tr><td rowspan="6" width="154"><p><strong>Negative</strong></p><br><br><p><strong>Control Group</strong></p></td><td width="468"><p>pABaC +gal1+mCherry</p></td></tr><tr><td width="468"><p>pCiRbS +gal1+mCherry</p></td></tr><tr><td width="468"><p>pbCiRS +gal1+mCherry</p></td></tr><tr><td width="468"><p>pABaC +gal1+EYFP</p></td></tr><tr><td width="468"><p>pbCiRS +gal1+EYFP</p></td></tr><tr><td width="468"><p>pCiRbS +gal1+EYFP</p></td></tr>
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                                             <tr><tr><td rowspan="4" width="154"><p><strong>Experimental</strong></p><br><br><p><strong>Group</strong></p></td><td width="468"><p>pABaC +pCiRbS +p1m</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS +p1m</p></td></tr><tr><td width="468"><p>pABaC +pCiRbS+p1E</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS +p1E</p></td></tr><tr><td rowspan="6" width="154"><p><strong>Negative</strong></p><br><br><p><strong>Control Group</strong></p></td><td width="468"><p>pABaC +p1m</p></td></tr><tr><td width="468"><p>pCiRbS +p1m</p></td></tr><tr><td width="468"><p>pbCiRS +p1m</p></td></tr><tr><td width="468"><p>pABaC +p1E</p></td></tr><tr><td width="468"><p>pbCiRS +p1E</p></td></tr><tr><td width="468"><p>pCiRbS p1E</p></td></tr>
 
                                         </tbody>
 
                                         </tbody>
 
                                     </table>
 
                                     </table>
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                                         </thead>
 
                                         </thead>
 
                                         <tbody>
 
                                         <tbody>
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                                             <tr><td width="154"><p><strong>Experimental</strong></p><p><strong>Group</strong></p></td><td width="468"><p>pABaC +pCiRbS +gal1+Fluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +pbCiRS +gal1+Fluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +pCiRbS +gal2+Nanoluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +pbCiRS +gal2+Nanoluc</p></td></tr><tr><td width="154"><p><strong>Negative</strong></p><p><strong>Control Group</strong></p></td><td width="468"><p>pABaC +gal1+Fluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pbCiRS +gal1+Fluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pCiRbS +gal1+Fluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +gal2+Nanoluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pbCiRS +gal2+Nanoluc</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pCiRbS +gal2+Nanoluc</p></td></tr>
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                                             <tr><td width="154"><p><strong>Experimental</strong></p><p><strong>Group</strong></p></td><td width="468"><p>pABaC +pCiRbS +p1F</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +pbCiRS +p1F</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +pCiRbS +p2N</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +pbCiRS +p2N</p></td></tr><tr><td width="154"><p><strong>Negative</strong></p><p><strong>Control Group</strong></p></td><td width="468"><p>pABaC +p1F</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pbCiRS +p1F</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pCiRbS +p1F</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pABaC +p2N</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pbCiRS +p2N</p></td></tr><tr><td width="154"><p><strong>&nbsp;</strong></p></td><td width="468"><p>pCiRbS +p2N</p></td></tr>
 
                                         </tbody>
 
                                         </tbody>
 
                                     </table>
 
                                     </table>
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                                         </thead>
 
                                         </thead>
 
                                         <tbody>
 
                                         <tbody>
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                                             <tr><td rowspan="4" width="154"><p><strong>Experimental</strong></p><p><strong>Group</strong></p></td><td width="468"><p>pABaC +pCiRbS +gal1+Nanoluc</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS +gal1+Nanoluc</p></td></tr><tr><td width="468"><p>pABaC +pCiRbS +gal2+Nanoluc</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS 1+gal2+Nanoluc</p></td></tr><tr><td rowspan="4" width="154"><p><strong>Control Group</strong></p></td><td width="468"><p>pABaC +gal2+Nanoluc</p></td></tr><tr><td width="468"><p>pbCiRS +gal2+Nanoluc</p></td></tr><tr><td width="468"><p>pCiRbS +gal2+Nanoluc</p></td></tr><tr><td width="468"><p>TDH3P+NanoLuc</p></td></tr>
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                                             <tr><td rowspan="4" width="154"><p><strong>Experimental</strong></p><p><strong>Group</strong></p></td><td width="468"><p>pABaC +pCiRbS +p1N</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS +p1N</p></td></tr><tr><td width="468"><p>pABaC +pCiRbS +p2N</p></td></tr><tr><td width="468"><p>pABaC +pbCiRS 1+p2N</p></td></tr><tr><td rowspan="4" width="154"><p><strong>Control Group</strong></p></td><td width="468"><p>pABaC +p2N</p></td></tr><tr><td width="468"><p>pbCiRS +p2N</p></td></tr><tr><td width="468"><p>pCiRbS +p2N</p></td></tr><tr><td width="468"><p>TDH3P+NanoLuc</p></td></tr>
 
                                         </tbody>
 
                                         </tbody>
 
                                     </table>
 
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                                     <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Tianjin--EYFP123.jpg">
 
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                                     <p>Figure 6. pABaC +pCiRbS+gal1+EYFP photoed by normal microscope and Leica DMi8 </p>
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                                     <p>Figure 6. pABaC +pCiRbS+p1E photoed by normal microscope and Leica DMi8 </p>
 
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                                Measuement result
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                                    <p>Figure 7. Experimental result reported by pABaC+pbCiRS+gal1+Fluc</p>
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                                    <p>Figure 8. Experimental result reported by pABaC+pCiRbS+gal1+Fluc</p>
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                                    <p>Figure 9. Experimental result reported by pABaC+pbCiRS+gal1+Nluc<br>(obtained by live cell assays)</p>
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                                    <p>Figure 10.Experimental result reported by pABaC+pCiRbS+gal1+Nluc<br>(obtained by live cell assays)</p>
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                                    <p>Figure 11.Experimental result reported by pABaC+pbCiRS+gal2+Nluc<br>(obtained by live cell assays)</p>
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                                    <p>Figure 12.Experimental result reported by pABaC+pCiRbS+gal2+Nluc<br>(obtained by live cell assays)</p>
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                                    <p>Some basic information can be gained from the above figures:</p>
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                                        <li>Compared with the corresponding control groups, all the experimental groups have showed apparent oscillatory signals.</li>
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                                        <li>In the measured 3500 min, the oscillations in 700-2000 min are extremely unstable and nearly absent, while the oscillations for the rest of the time are relatively stable.</li>
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                                        <li>The cycle is about 500min (about 8.3h).</li>
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                                    <p><strong><strong>Note: final measurement results and analysis should be accessed in <a href="https://2018.igem.org/Team:Tianjin/Demonstrate">Demonstrate</a> page.</strong></strong></p>
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MEASUREMENT

In pursuit of a rigorously scientific result, we have adopted multiple measuring approaches throughout the whole project, among which two predominant methods described in extreme detail in wiki, would inspire relative researchers.

As two sophisticated and renowned reporters in biotechnological domain, fluorescent protein and luciferase were opted to illustrate the effectiveness of our constructed oscillating system, detected by combined means and instrument. Mcherry and EYFP, two selected fluorescent reporters were measured by both fluorescence spectrophotometer and Leica DMi8 respectively. Leica DMi8 allows us to visually and intuitively see the expression of the fluorescent protein EYFP in Saccharomyces cerevisiae In terms of luciferase, in addition to commonly used firefly luciferase, a courageous attempt we made was to choose a novel luciferase NanoLuc and its live cell substrate in spite of no records of the usefulness in Saccharomyces cerevisiae before and obtained expected outcome successfully.

How did we choose our reporter gene?
How did we design our measurement plan?