Difference between revisions of "Team:Jilin China/Demonstrate"

 
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<section class="s1">
 
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   <p><span>TOOLKITS</span><br>VERSION 3.0</p>
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   <p><span>Project</span><br>Demonstration</p>
 
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     <td><a href="#pragraph_1" class="clickwave">Promoters</a></td>
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     <td><a href="#pragraph_1" class="clickwave">Demonstrate</a></td>
 
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  <div class="title_nav"><h2>VERSION 3.0</h2></div>
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  <div class="title_nav"><h2>Demonstrate</h2></div>
 
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   <li><a href="#pragraph_1">Promoters</a></li>
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   <li><a href="#pragraph_1">Demonstrate</a></li>
 
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      <h2>Results</h2>
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<h2>Demonstrate</h2>
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<p>We added 10 cold-reprssible RNA-based thermosensors and 8 cold-inducible RNA-based thermosensors to the SynRT Toolkit. After that, we named the updated toolkit as SynRT Toolkit version 3.0.</p>
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<h4 class="tables"><b>·RNA-based thermosensors can sense different temperatures</b></h4>
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<h3>Cold-repressible RNA-based thermosensors</h3>
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<p>We designed four different types of RNA-based thermosensors, including heat-inducible RNA-based thermosensor, heat-repressible RNA-based thermosensor, cold-inducible RNA-based thermosensor and cold-repressible RNA-based thermosensor. We have designed more than 200 different thermosensors, and constructed their measurement device. </p>
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<p>Cold-repressible RNA-based thermosensors are designed based on the RNase III. We constructed their measurement devices and measured the activities at five temperatures: 15, 25, 29, 35 and 37℃. The measured temperature range is lower than the heat-inducible RNA-based thermosensors' temperature range. We also designed a new negtive control, which always has a cleavage site of RNase III, so it will be digest by the enzyme. After measurement, we chose 10 out of 50 thermosensors. </p>
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<p>After the experiments, we got some inspiring results. As <b>figure 1</b> shows, heat-inducible RNA-based thermosensors' activities increase at elevated temperature. Heat-repressible RNA-based thermosensors' activities decrease with the increasement of temperature. Besides, cold-inducibe RNA-based thermosensors show lower sensing temperature range than heat-repressible RNA thermosensor, whose intensity decrease sharply from 15 to 20℃. And cold-repressible RNA-based thermosensors' activity decrease with decreasing temperature even below 29℃.</p>
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<p>Figure 1 shows the change of activities of different thermosensors. We find that all of these thermosensors' activities decrease with temperature decreasing. </p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/29/T--Jilin_China--demonstrate--hot.png" width="75%"/></div>
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<img src="https://static.igem.org/mediawiki/2018/4/48/T--Jilin_China--result--R3bar.png" width="95%" />
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<p class="figure">Figure 1. Heat map of four different types of RNA-based thermosensors. Rows represent activity levels of different thermosensors. These values are normalized using the Fluorescence/Abs600 of positive control. (A) includes 48 heat-inducible RNA-based thermosensors' activities at 29, 31, 35, 37, 39 and 42℃. (B) includes 22 heat-repressible RNA-based thermosensors' activities at 29, 37 and 42℃. (C) includes 10 cold-repressible RNA-based thermosensors' activities at 15, 25, 29, 35 and 37℃. (D) includes 8 cold-inducible RNA-based thermosensors' activities at 15, 20, 25℃.</p>
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<p>For most of the heat-inducible RNA-based thermosensors, the sensing temperature range is between 30 to 40. However, for the cold-repressible RNA-based thermosensors, the sensing temperature range decrease to 15~35℃.</p>
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<img src="https://static.igem.org/mediawiki/2018/7/7c/T--Jilin_China--result--R3fold.png" width="95%" />
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<h4 class="tables"><b>·Features of RNA-based thermosensors can be computed by using fitted curve</b></h4>
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    <p>Discussion: As these results show, cold-repressible RNA-based thermosensors work really well. Their activities decrease with the temperature decreasing. And the sensing temperature range is different from heat-inducible RNA-based thermosensors'. Cold repressible RNA-based thermosensor can sense lower temperature. </p>
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<p>In order to get the melting temperature, intensity and sensitivity of the thermosensors, we fitted a curve to reflect the relationship between the change of temperature and the expression intensity of thermosensors (Figure 2). </p>
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  <h3>Cold-inducible RNA-based thermosensors</h3>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/2/2e/T--Jilin_China--demon--29.jpeg" / width="60%"></div>
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  <p>Cold-inducible RNA-based thermosensors are designed based on the cspA 5'UTR mRNA, by changing psedoknot length, GC content, Wobble base pair, we designed 50 different thermosensors, and measured their activity in 15, 20 and 25℃. According to the result, we screened 8 out of 50 thermosensors. </p>
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<p class="figure">Figure 2. The fitted curve of BBa_K2541029. The black dash line is the tangent line at melting temperature. The intersection of the upper gray dash line and curve represents the stem-loop structures of thermosensors are all destroyed. The intersection of the medial gray dash line and curve represents a 50% switch in expression occurs, and it is defined as melting temperature. The intersection of lower gray dash line and curve represents the stem-loop structure of thermosensors all exsit.</p>
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  <p>As the figure 3 shows, all the thermosensors' activities decrease at elevated temperature. </p>
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<br>
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  <img src="https://static.igem.org/mediawiki/2018/0/0f/T--Jilin_China--result--cspabar2.png" width="95%" />
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<br>
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  <p>The sensing temperature range of cold-inducible RNA-based thermosensors is lower than heat-repressive RNA-based thermosensors. As the figure 4 shows, most of the cold-inducible RNA-based thermosensors' activity decrease sharply from 15 to 20℃. After that, the change of activity gradually levels off. </p>
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<p>Table 1 shows the features of all the heat-inducible thermosensors we obtained from the fitting curve.</p>
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  <img src="https://static.igem.org/mediawiki/2018/8/84/T--Jilin_China--result--cspafold.png" width="95%" />
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<p class="figure">Table 1. Features of the heat-inducible RNA-based thermosensors</p>
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<p>As these results show, cold-induced RNA-based thermosensors can work. Their activities decrease at elevated temperature. And the sensing temperature range is lower than heat-repressible RNA-based thermosensors'.</p>
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<div align="center"><img src="https://static.igem.org/mediawiki/2018/f/fe/T--Jilin_China--demon--biao.png" width="50%"/></div>
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<p><b>To sum up: Our SynRT toolkit version 3.0, including heat-inducible RNA thermosensors, heat-repressible RNA-based thermosensors, cold-inducible RNA-based thermosensors and cold-repressible RNA-based thermosensors can work under realistic conditions. You can read more in our <a href="https://2018.igem.org/Team:Jilin_China/Demonstrate">Demonstrate page</a>.</b></p>
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<p>These features point to the diversity in thermosensor response. These thermosensors' melting temperatures range 33 to 55℃, with different relative intensity and sensitivity.</p>
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<h4 class="tables">·SynRT toolkit is developed and updated to version 3.0 </h4>
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<p>Based on these data, we classified these thermosensors by melting temperature, relative intensity and sensitivity, and developed a search engine -- SynRT Explorer. You can visit our <a href="https://2018.igem.org/Team:Jilin_China/Part/Search_Engine">Search Engine page</a> to use it. </p>
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Latest revision as of 02:30, 8 December 2018

Project
Demonstration


Demonstrate

Demonstrate

  • Demonstrate

    ·RNA-based thermosensors can sense different temperatures

    We designed four different types of RNA-based thermosensors, including heat-inducible RNA-based thermosensor, heat-repressible RNA-based thermosensor, cold-inducible RNA-based thermosensor and cold-repressible RNA-based thermosensor. We have designed more than 200 different thermosensors, and constructed their measurement device.

    After the experiments, we got some inspiring results. As figure 1 shows, heat-inducible RNA-based thermosensors' activities increase at elevated temperature. Heat-repressible RNA-based thermosensors' activities decrease with the increasement of temperature. Besides, cold-inducibe RNA-based thermosensors show lower sensing temperature range than heat-repressible RNA thermosensor, whose intensity decrease sharply from 15 to 20℃. And cold-repressible RNA-based thermosensors' activity decrease with decreasing temperature even below 29℃.

    Figure 1. Heat map of four different types of RNA-based thermosensors. Rows represent activity levels of different thermosensors. These values are normalized using the Fluorescence/Abs600 of positive control. (A) includes 48 heat-inducible RNA-based thermosensors' activities at 29, 31, 35, 37, 39 and 42℃. (B) includes 22 heat-repressible RNA-based thermosensors' activities at 29, 37 and 42℃. (C) includes 10 cold-repressible RNA-based thermosensors' activities at 15, 25, 29, 35 and 37℃. (D) includes 8 cold-inducible RNA-based thermosensors' activities at 15, 20, 25℃.

    ·Features of RNA-based thermosensors can be computed by using fitted curve

    In order to get the melting temperature, intensity and sensitivity of the thermosensors, we fitted a curve to reflect the relationship between the change of temperature and the expression intensity of thermosensors (Figure 2).

    Figure 2. The fitted curve of BBa_K2541029. The black dash line is the tangent line at melting temperature. The intersection of the upper gray dash line and curve represents the stem-loop structures of thermosensors are all destroyed. The intersection of the medial gray dash line and curve represents a 50% switch in expression occurs, and it is defined as melting temperature. The intersection of lower gray dash line and curve represents the stem-loop structure of thermosensors all exsit.



    Table 1 shows the features of all the heat-inducible thermosensors we obtained from the fitting curve.

    Table 1. Features of the heat-inducible RNA-based thermosensors

    These features point to the diversity in thermosensor response. These thermosensors' melting temperatures range 33 to 55℃, with different relative intensity and sensitivity.

    ·SynRT toolkit is developed and updated to version 3.0

    Based on these data, we classified these thermosensors by melting temperature, relative intensity and sensitivity, and developed a search engine -- SynRT Explorer. You can visit our Search Engine page to use it.