Difference between revisions of "Team:Tianjin/Measurement"

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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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                                        <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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                                              <th colspan="12">Table 2. Chosen luciferase</th>
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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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                                    <p>Figure 2. Mechanism of luciferase</p>
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                                    <p>Figure 3.The bioluminescent reaction catalyzed by Fluc and Nluc</p>
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                                How did we design our measurement plan?
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                                        <p>Clock Mechanisms II:Post-translational Feedback Loops</p>
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                                    <p>
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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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                                              <th colspan="12">Table3.1 day’s measurement ----- Fluorescent protein detection</th>
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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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                                        </tbody>
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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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                                <div class="col-xs-12 picture">
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                                    <img src="https://static.igem.org/mediawiki/2018/8/8a/T--Tianjin--measurement5.jpg">
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                                    <p>Figure 5. Procedures for measuring luciferase via luminaskan microplate reader</p>
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                                    <p>
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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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                                              <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>
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                                        </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>
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                                        </tbody>
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                                    </table>
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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>
 +
                                <div class="col-xs-12 text">
 +
                                    <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.
 +
                                    </p>
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                                    <img src="https://static.igem.org/mediawiki/2018/3/3e/T--Tianjin--measurement6.jpg">
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                                    <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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                                <div class="col-xs-12 picture">
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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>
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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">
 +
                                    <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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                                Measuement instrument
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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>
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                        <br>
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                    </p>
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                    <p class="reftext" id="re2">
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                        <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>
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                    <p class="reftext" id="re3">
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                        <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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                        <br>
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<p>There are a lot of exciting parts in the Registry, but many parts have still not been characterized. Synthetic Biology needs great measurement approaches for characterizing new parts, and efficient new methods for characterizing many parts at once. If you've done something exciting in the area of Measurement, describe it here!</p>
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<p>If you've done excellent work in measurement, you should consider nominating your team for this special prize. Designing great measurement approaches for characterizing new parts or developing and implementing an efficient new method for characterizing thousands of parts are good examples.
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To compete for the <a href="https://2018.igem.org/Judging/Awards">Best Innovation in Measurement prize</a>, please describe your work on this page and also fill out the description on the <a href="https://2018.igem.org/Judging/Judging_Form">judging form</a>.
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<p>You can look at what other teams did to get some inspiration! <br />
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Here are a few examples:</p>
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                        BEIYANG CAMPUS,TIANJIN UNIVERSITY,TIANJIN
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<li><a href="https://2016.igem.org/Team:Stanford-Brown">2016 Stanford-Brown</a></li>
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<li><a href="https://2016.igem.org/Team:Genspace">2016 Genspace</a></li>
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<li><a href="https://2015.igem.org/Team:William_and_Mary">2015 William and Mary</a></li>
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<li><a href="https://2014.igem.org/Team:Aachen">2014 Aachen  </a></li>
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Revision as of 17:55, 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