Difference between revisions of "Team:Jilin China/Result/Version 1"

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   <p><span>TOOLKIT</span><br>VERSION 1.0</p>
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   <p><span>Project</span><br>Demonstration</p>
 
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     <td><a href="#paragraph_1" class="clickwave">Results</a></td>
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  <div class="title_nav"><h2>VERSION 1.0</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>SynRT toolkit version 1.0 is the first generation thermosensors collection we provide to users. It contains 48 heat-induced RNA-based thermosensors. </p>
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<h4 class="tables"><b>·RNA-based thermosensors can achieve temperature sensing</b></h4>
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<p>We constructed in a total of 150 heat-inducible RNA thermosensors using goldengate assembly described in the <a href="https://2018.igem.org/Team:Jilin_China/Construction">Construction page</a>. And the measurement method was describled in <a href="https://2018.igem.org/Team:Jilin_China/Construction">Measurement page</a>. </p>
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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>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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<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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<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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<h4 class="tables"><b>·Activities of thermosensors increase at elevated temperature</b></h4>
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<h4 class="tables"><b>·Features of RNA-based thermosensors can be computed using fitted curve</b></h4>
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<p>We measured the activities of these thermosensors at six different temperatures: 29, 31, 35, 37, 39 and 42℃, and then fitted curve. Through the curve fitting, we discarded some thermosensors, which cannot fit the curve or have too high or too low melting temperature. Upon screening, we selected 48 heat-inducible RNA thermosensors out of the 150 members. And <b>figure 1</b> shows the 48 thermosensors' activity. The positive control we designed won't form stem-loop structure with SD sequence by software prediction, and the experimental charaterization is described in <a href="https://2018.igem.org/Team:Jilin_China/Measurement">Measurement page</a>. The normalized fluorescence are used to describe the change of activity compared with positive control. We find that all of these thermosensors' expression level increase at elevated temperature, and the extent of increase relative to temperature is different. </p>
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<p>In order to make users select a thermosensor conveniently by getting 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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<img src="https://static.igem.org/mediawiki/2018/2/2e/T--Jilin_China--demon--29.jpeg" />
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<img src="https://static.igem.org/mediawiki/2018/2/2e/T--Jilin_China--result-rtbar.png" width="95%" length="95%"></img>
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<center>Figure 2. The fitted curve of BBa_K2541029. The blak 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 curcve represents the stem-loop structure of thermosensors all exsit.</center>
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<p>To quantify different features of this library, we computed the fold-change of the response in the given temperature range. As the figure shows, from 29 to 37℃, the fold change ranges from 2.26 fold to 7.17 fold. And from 37 to 42℃, the fold change ranges from 1.40 fold to 3.01 fold. These fold-changes were higher than positive control.  These results show that there is a diverse set of heat-inducible RNA thermosensors in toolkit.</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/2018/6/6f/T--Jilin_China--result--rtfold.png" width="95%" length="95%"></img>
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<center>Table 1. Features of the heat-inducible RNA-based thermosensors</center>
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<p>Conclusion: as these results show, the heat-inducible RNA thermosensor we designed can work. The fluorescence value increases with temperature elevated. Besides, the difference in fluorescence intensity and the rate of increase points to the diversity in thermosensor response. We think the sequence change in stem length, loop size, and mismatched or bulges in the stem give rise to the diversity in thermosensor response.</p>
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<img src="https://static.igem.org/mediawiki/2018/f/fe/T--Jilin_China--demon--biao.png" />
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<p>These features points to the diversity in thermosensor response. These thermosensors' melting temperature ranges 33 to 55℃, with different relative intensity and sensitivity.</p>
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    <h4 class="tables"><b>·Thermosensors' features can be computed by fitting curve</b></h4>
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    <p>Combined with our human practice work we have mentioned in the <a href="">Overview page</a>. We also have built switching behavior fitting model to describe the continuous switching behavior of thermosenors.</p>
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<h4 class="tables">·SynRT toolkit is developed and updated to version 3.0 </h4>
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<center><a href="https://2018.igem.org/Team:Jilin_China/Model">>For more details about our model, you can visit Model page.<</a></center>
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<p>Based on these datas, we classified these thermosensors, and develop 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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<p>From the fitted curve, we can get the melting temperature, relative intensity and sensitivity of thermosensors.  
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<p>You can see the individual result as follows:</p>
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<center>Figure 3. (A) Experiment measurement of the individual heat-inducible RNA-based thermosensor. The height of the bars represent the normalized fluorescence, which is the mean of three replication. The bar colors purplr, aquamarine, light green, orange, red and brown represent the temperature 29, 31, 35, 37, 39 and 42℃. (B) The fitted curve of individual thermosensor, the black dash line is the tangent line at the melting temperature. The intersection of the upper gray dash line and curve represents the stem-loop structures are all destroyed. The intersection of the medial gray dash line and curve represents a 50% switch in expression occurs. The intersection of the lower gray dash line and curve represents the stem-loop structures all exist.  (C) The features of thermosensors computed through the fitted curve. Melting tempurature is the temperature at which a 50% switch in expression occurs. Sensitivity is defined as the value of derivative at melting temperature. Relative intensity is the predicted intensity when all of the stem-loop structure in thermosensor mRNA were destroyed.</center>
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<p><b>Conclusion:</b> Through the fitting curve, we can compute the features of our thermosensors. Based on these features, we conclude that thermosensors have different sensing range, most of them are from 35 to 37℃. Additionally, they also have some difference in activity and sensitivity. Due to we have these diverse set of heat-inducible RNA-based thermosensors, we classified them by melting temperature, relative intensity and sensitivity. Based on these datas, we classified these thermosensors, and develop 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></p>
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Revision as of 15:42, 15 October 2018

Project
Demonstration


Demonstrate

Demonstrate

  • Demonstrate

    ·RNA-based thermosensors 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℃.

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

    In order to make users select a thermosensor conveniently by getting 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 blak 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 curcve 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 points to the diversity in thermosensor response. These thermosensors' melting temperature ranges 33 to 55℃, with different relative intensity and sensitivity.

    ·SynRT toolkit is developed and updated to version 3.0

    Based on these datas, we classified these thermosensors, and develop a search engine -- SynRT Explorer. You can visit our Search Engine page to use it.