Difference between revisions of "Team:Tianjin/Results"

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                    JUDGING FORM             
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                    <p>Measurment</p>
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                                How did we choose our reporter gene?
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                                    <p>
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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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                                        <p>Fluorescent protein</p>
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                                    <p>
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                                      In search for the part kit provided by the iGEM headquarter, we found several available fluorescent proteins. Under the instruction of our <a href="https://2018.igem.org/Team:Tianjin/Model">fluorescent protein judging model</a>, we finally chose mCherry and EYFP as our fluorescent protein reporter.
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                                              <th colspan="12">Table 1. Chosen fluorescent proteins</th>
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                                            <tr><td><p>Protein</p></td><td>λ<sub>em</sub>(nm)</td><td>λ<sub>em</sub>(nm)</td><td><p>part serial number</p></td><td><p>site</p></td></tr><tr><td><p>mCherry</p></td><td><p>587</p></td><td><p>610</p></td><td><p>Part:BBa_E2060</p></td><td><p>3-7L</p></td></tr><tr><td><p>EYFP</p></td><td><p>513</p></td><td><p>527</p></td><td><p>Part:BBa_E2030</p></td><td><p>3-7J</p></td></tr>
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                                    <p>Figure 1. Mechanism of fluorescent proteins</p>
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<h1>Results</h1>
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<p>Here you can describe the results of your project and your future plans. </p>
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                                        <p>Luciferase</p>
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                                    <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>
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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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                                    </p>
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                                              <th colspan="12">Table 2. Chosen luciferase</th>
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                                            </tr>
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                                        </thead>
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                                        <tbody>
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                                            <tr><td width="113"><p>luciferase</p></td><td width="113">λ<sub>em</sub>(nm)</td><td width="174"><p>resource</p></td><td width="200"><p>Detail</p></td></tr><tr><td width="113"><p>Fluc</p></td><td width="113"><p>560</p></td><td width="174"><p>Bought from Promega</p></td><td width="200"><p>pGL4 Luciferase Reporter Vectors</p></td></tr><tr><td width="113"><p>Nluc</p></td><td width="113"><p>460</p></td><td width="174"><p>Part:BBa_K1680009</p></td><td width="200"><p>Kit site:7-3N</p></td></tr>
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                                    <img src="https://static.igem.org/mediawiki/2018/2/27/T--Tianjin--measurement2.jpg">
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                                    <p>Figure 2. Mechanism of luciferase</p>
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                                    <img src="https://static.igem.org/mediawiki/2018/c/cd/T--Tianjin--measurement3.jpg">
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                                    <p>Figure 3.The bioluminescent reaction catalyzed by Fluc and Nluc</p>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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                                How did we design our measurement plan?
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<h3>Describe what your results mean </h3>
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                                        <p>Clock Mechanisms II:Post-translational Feedback Loops</p>
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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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>
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                                      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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<h3> Project Achievements </h3>
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                                              <th colspan="12">Table3.1 day’s measurement ----- Fluorescent protein detection</th>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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<ul>
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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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<li>A list of linked bullet points of the successful results during your project</li>
+
                                        </tbody>
−
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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                                    <img src="https://static.igem.org/mediawiki/2018/4/4a/T--Tianjin--measurement4.jpg">
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                                    <p>Figure 4. Procedures for measuring mCherry/EYFP via fluorescent spectrophotometer</p>
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                                    <img src="https://static.igem.org/mediawiki/2018/8/8a/T--Tianjin--measurement5.jpg">
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<div class="highlight decoration_A_full">
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                                    <p>Figure 5. Procedures for measuring luciferase via luminaskan microplate reader</p>
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<h3>Inspiration</h3>
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                                </div>
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<p>See how other teams presented their results.</p>
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<ul>
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                                    <p>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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                                        Detailed methods are available in our<a href="https://2018.igem.org/Team:Tianjin/Experiments"> Experiment</a>.<br><br>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
                                    </p>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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                                              <th colspan="12">Table 4.2 days’ measurement ----- Fluc&Nluc detection in lytic cells</th>
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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>
 +
                                        </tbody>
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                                    </table>
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                                              <th colspan="12">Table 5.3 days’ measurement ----- Nluc detection in live cells</th>
 +
                                            </tr>
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                                        </thead>
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                                        <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>
 +
                                        </tbody>
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                                    <div class="title title-normal">
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                                        <p>EYFP measurement using Leica DMi8</p>
 +
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                                </div>
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                                <div class="col-xs-12 text">
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                                    <p>
 +
                                        Leica DMi8 is an excellent inverted microscope for life science research, which is utilized extensively in materials inspection and measurement. For live cell research, the DMi8 platform is a complete solution whether precisely follow the development of a single cell in a dish, screen through multiple assays, obtain single molecule resolution, or tease out behaviors of complex processes. <br>
 +
                                        Combined with easy to use software, high resolution cameras, and brilliant LED illumination, these inverted research systems are flexible to see the expression of the fluorescent protein EYFP in <em>Saccharomyces cerevisiae</em>. Through high speed imaging, using the external fluorescent filter wheels, we obtained fast and accurate fluorescent images as following show. It tells that our bacteria that we constructed express EYFP successfully and continuously in oscillating system. Meanwhile, the fluorescence intensity detected was very bright, indicating that the expression of EYFP was at a high level.
 +
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                                    <img src="https://static.igem.org/mediawiki/2018/3/3e/T--Tianjin--measurement6.jpg">
 +
                                    <p>Figure 6. pABaC +pCiRbS+gal1+EYFP photoed by Leica DMi8</p>
 +
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                                Measuement result
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                                    <img src="https://static.igem.org/mediawiki/2018/1/12/T--Tianjin--measurement7.jpg">
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                                    <p>Figure 7. Experimental result reported by pABaC+pbCiRS+gal1+Fluc</p>
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                                    <img src="https://static.igem.org/mediawiki/2018/4/45/T--Tianjin--measurement8.jpg">
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                                    <p>Figure 8. Experimental result reported by pABaC+pCiRbS+gal1+Fluc</p>
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                                <div class="col-xs-12 picture">
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                                    <img src="https://2018.igem.org/File:T--Tianjin--measurement9.jpg">
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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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                                <div class="col-xs-12 picture">
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                                    <img src="https://static.igem.org/mediawiki/2018/3/36/T--Tianjin--measurement10.jpg">
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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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                                <div class="col-xs-12 picture">
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                                    <img src="https://static.igem.org/mediawiki/2018/d/d6/T--Tianjin--measurement11.jpg">
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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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                                <div class="col-xs-12 picture">
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                                    <img src="https://static.igem.org/mediawiki/2018/7/7a/T--Tianjin--measurement12.jpg">
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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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                                <div class="col-xs-12 text">
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                                    <p>Some basic information can be gained from the above figures:</p>
 +
                                    <ol>
 +
                                        <li>Compared with the corresponding control groups, all the experimental groups have showed apparent oscillatory signals.</li>
 +
                                        <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>
 +
                                        <li>The cycle is about 500min (about 8.3h).</li>
 +
                                    </ol>
 +
                                    <p><strong><strong>Note: final measurement results and analysis should be accessed in <a href="https://2018.igem.org/Team:Tianjin/Demonstrate">Result</a> page.</strong></strong></p>
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                                    <p>
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                                      Fluorescent protein: <br>
 +
                                      Hitachi F-2500 Flourescence Spectrophotometer;<br>
 +
                                      Leica DMi8 inverted microscope<br>
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                                      Luciferase : Thermoscientific Luminoskan Microplate Reader<br>
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                    <h1>References</h1>
 +
                    <p class="reftext" id="re1">
 +
                        <a>[1]Ow, D.W. et al. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and trans-genic plants. Science 234, 856–9.</a>
 +
                        <br>
 +
                    </p>
 +
                    <p class="reftext" id="re2">
 +
                        <a>[2]De Wet, J.R. et al. (1987) Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–37.</a>
 +
                        <br>
 +
                    </p>
 +
                    <p class="reftext" id="re3">
 +
                        <a>[3]Chen, AH (Chen, Anna H.); Lubkowicz,D (Lubkowicz, David); Yeong, V (Yeong, Vivian); Chang, RL (Chang, Roger L.); Silver, PA(Silver, Pamela A.)  Transplantability of a circadian clock to a noncircadian organism. SCIENCE ADVANCES(2015) DOI: 10.1126/sciadv.1500358</a>
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                        BEIYANG CAMPUS,TIANJIN UNIVERSITY,TIANJIN
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Latest revision as of 18:15, 13 October 2018

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Measurment

How did we choose our reporter gene?
How did we design our measurement plan?
Measuement result
Measuement instrument