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

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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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      <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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<h3 class="tables">·RNA-based thermosensor can achieve temperature sensing</h3>
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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 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 inspired results. As <b>figure 1</b> shows, heat-inducible RNA-based thermosensors' activities increase at elevated temperature. Heat-repressible RNA-based thermosensors' activity decrease with increasement of temperature. Besides, cold-inducibe RNA-based thermosensors show lower sensing temperature range than heat-repressible RNA thermosensor, their 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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<img src="https://static.igem.org/mediawiki/2018/2/29/T--Jilin_China--demonstrate--hot.png" />
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<center>Figure 1. Heat map of four different types of RNA-based thermosensors. Rows represent activity levels of different thermosensors. The activity levels are the mean of three replications. 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 23 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℃.</center>
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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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    <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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  <h3>Cold-inducible RNA-based thermosensors</h3>
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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>As the figure 3 shows, all the thermosensors' activities decrease at elevated temperature. </p>
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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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  <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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  <img src="https://static.igem.org/mediawiki/2018/8/84/T--Jilin_China--result--cspafold.png" width="95%" />
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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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<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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Revision as of 10:48, 15 October 2018

Project
Demonstration


Demonstrate

Demonstrate

  • Demonstrate

    ·RNA-based thermosensor can achieve temperature sensing

    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 designed more than 200 different thermosensors, and constructed their measurement device.

    After the experiments, we got some inspired results. As figure 1 shows, heat-inducible RNA-based thermosensors' activities increase at elevated temperature. Heat-repressible RNA-based thermosensors' activity decrease with increasement of temperature. Besides, cold-inducibe RNA-based thermosensors show lower sensing temperature range than heat-repressible RNA thermosensor, their 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. The activity levels are the mean of three replications. 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 23 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℃.