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

Line 74: Line 74:
 
     <li class="pragraph_1 start" id="pragraph_1">
 
     <li class="pragraph_1 start" id="pragraph_1">
 
     <div>
 
     <div>
−
       <h2>aaa</h2>
+
       <h2>Overview</h2>
−
      <p></p>
+
<p>After designing a large number of RNA thermosensors, we began to think how to measure these thermosensors to get their melting temperature, intensity and sensitivity. After reading a large amount of relevant literature, we designed the following measurement device, which consists of a promoter, RNA thermosensor, sfGFP and double terminator. We have added different type of measurement devices to the parts registry. </p>
 +
<p>The measurement protocol has been added to the Protocol page, you can click here to read. And for more information about the results, you can visit our results page.</p>
 
     </div>
 
     </div>
 
     </li>
 
     </li>
 
     <li class="pragraph_2" id="pragraph_2">
 
     <li class="pragraph_2" id="pragraph_2">
 
     <div>
 
     <div>
−
       <h2>aaa</h2>
+
       <h2>Construction Device</h2>
−
       <p></p>
+
<h3>1. promoter</h3>
 +
       <p>In the selection of promoters, we have did pre-experiments to select suitable promoter for different types of RNA thermosensors, ensured that the intensity difference between different RNA thermosensors can be measured, and avoid the inaccurate. </p>
 +
<h3>2. RNA thermosensor</h3>
 +
<p>RNA thermosensor is the temperature sensing element and the site of ribosome binding. We have designed four different types of RNA thermosensor. For more information about the design of the RNA thermosensor, you can visit our Design page. Click Here!</p>
 +
<h3>3.  sfGFP</h3>
 +
<p>sfGFP, superfolder GFP，is the reporter protein of measurement device. It develops fluorescence about 3fold faster than mut3 GFP and reaches 4fold higher absolute fluorescence levels. Fluorescenct colonies can be identified with the naked eye even without UV or blue light illumination. Additionally it is more stable in vitro and refolds faster after in vitro denaturation with respect to mut3 GFP.</p>
 +
<p>Since we used the Goldengate assembly this year, the sfGFP from iGEM parts registry cannot be used because it contains the commonly used IIS restriction enzyme site. In order to solve this problem, we designed BbsI free site-directed mutagenesis sfGFP for goldengate and prokaryotic codon-optimism sfGFP_optimism. We compared these two parts with sfGFP and added them to the registry. As can be seen from yje data, sfGFP_optimism has a strong fluorescence intensity, so we finally use sfGFP_optimism in the construction device.</p>
 +
<p>For more information about the Goldengate assembly, you can visit the Construction page.</p>
 +
<center>Click Here!</center>
 +
<p>For more information about the improvement of sfGFP, you can visit the Improve page.</p>
 +
<center>Click Here!</center>
 +
<h3>4. Double terminator</h3>
 +
<p>In order to prevent the leakage of the gene, we decided to added two terminator downstream of the sfGFP.</p>
 +
    </div>
 +
    </li>
 +
    <li class="pragraph_3 start" id="pragraph_3">
 +
    <div>
 +
      <h2></h2>
 +
<p></p>
 +
<p></p>
 
     </div>
 
     </div>
 
     </li>
 
     </li>

Revision as of 09:33, 9 October 2018

MEASUREMENT


Promoters

Measurement

  • Overview

    After designing a large number of RNA thermosensors, we began to think how to measure these thermosensors to get their melting temperature, intensity and sensitivity. After reading a large amount of relevant literature, we designed the following measurement device, which consists of a promoter, RNA thermosensor, sfGFP and double terminator. We have added different type of measurement devices to the parts registry.

    The measurement protocol has been added to the Protocol page, you can click here to read. And for more information about the results, you can visit our results page.

  • Construction Device

    1. promoter

    In the selection of promoters, we have did pre-experiments to select suitable promoter for different types of RNA thermosensors, ensured that the intensity difference between different RNA thermosensors can be measured, and avoid the inaccurate.

    2. RNA thermosensor

    RNA thermosensor is the temperature sensing element and the site of ribosome binding. We have designed four different types of RNA thermosensor. For more information about the design of the RNA thermosensor, you can visit our Design page. Click Here!

    3. sfGFP

    sfGFP, superfolder GFP，is the reporter protein of measurement device. It develops fluorescence about 3fold faster than mut3 GFP and reaches 4fold higher absolute fluorescence levels. Fluorescenct colonies can be identified with the naked eye even without UV or blue light illumination. Additionally it is more stable in vitro and refolds faster after in vitro denaturation with respect to mut3 GFP.

    Since we used the Goldengate assembly this year, the sfGFP from iGEM parts registry cannot be used because it contains the commonly used IIS restriction enzyme site. In order to solve this problem, we designed BbsI free site-directed mutagenesis sfGFP for goldengate and prokaryotic codon-optimism sfGFP_optimism. We compared these two parts with sfGFP and added them to the registry. As can be seen from yje data, sfGFP_optimism has a strong fluorescence intensity, so we finally use sfGFP_optimism in the construction device.

    For more information about the Goldengate assembly, you can visit the Construction page.

    Click Here!

    For more information about the improvement of sfGFP, you can visit the Improve page.

    Click Here!

    4. Double terminator

    In order to prevent the leakage of the gene, we decided to added two terminator downstream of the sfGFP.