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

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     <div>
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       <h2>Construction Device</h2>
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       <h2>Measurement Device</h2>
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<h3>1. promoter</h3>
+
<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>
 
       <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>
 
<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>
 
<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>
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<h3>3. sfGFP</h3>
+
<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>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>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>
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<p>For more information about the Goldengate assembly, you can visit the Construction page.</p>
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<p>For more information about the Goldengate assembly, you can visit the Construction page. <b><a href="https://2018.igem.org/Team:Jilin_China/Construction">Click Here!</a></b></p>
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<center>Click Here!</center>
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<p>For more information about the improvement of sfGFP, you can visit the Improve page. <b><a href="https://2018.igem.org/Team:Jilin_China/Improve">Click Here!</a></b></p>
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<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>
 
<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>
 
<p>In order to prevent the leakage of the gene, we decided to added two terminator downstream of the sfGFP.</p>
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       <h2></h2>
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       <h2>Methods</h2>
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<p></p>
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<p>Before we did experiment, we encountered a problem -- what methods do we use to accurately reflect the change in our RNA thermosensor with temperature?</p>
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<p></p>
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<p>Before solving this problem, we must determine how the expression system of the chassis organism E.coli DH5α is affected by temperature. In order to solve this problem, we have designed positive control device wieh different intensity. Their RBS sequence predicted by the software will not form a stem-loop structure.</p>
 +
<p>Fudan_China team helped us measured some of the positive controls and plotted sfGFP expression intensity as a function of temperature and got the following results:</p>
 +
<p>As the figure shows that the intensity of expression of sfGFP by E.coli also increases at the elevated temperature. So we came to the following conclusions:</p>
 +
<li>1. E. coli DH5α protein expression system will be affected by temperature.</li>
 +
<li>2. The change of temperature and the expression level of sfGFP are approximately linear.</li>
 +
<p>We are very grateful to Fudan China for helping us in this part of the experiment. Other cooperation about us will be recorded on the Collaboration page, <a href="https://2018.igem.org/Team:Jilin_China/Collaborations">you can Click Here!</a></p>
 +
<p>After obtaining such a conclusion, it is important to eliminate the influence of temperature to the bacterial expression system. Therefore, as for the data processing, we mainly reflect the sfGFP intensity expressed by bacteria at a unit concentration by RFU. And the related RFU is used to reflect the change of expression intensity with different temperature in the experimental group compared with the control group. Their calculation methods are as follows:</p>
 
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Revision as of 18:32, 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.

  • Measurement 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.

  • Methods

    Before we did experiment, we encountered a problem -- what methods do we use to accurately reflect the change in our RNA thermosensor with temperature?

    Before solving this problem, we must determine how the expression system of the chassis organism E.coli DH5α is affected by temperature. In order to solve this problem, we have designed positive control device wieh different intensity. Their RBS sequence predicted by the software will not form a stem-loop structure.

    Fudan_China team helped us measured some of the positive controls and plotted sfGFP expression intensity as a function of temperature and got the following results:

    As the figure shows that the intensity of expression of sfGFP by E.coli also increases at the elevated temperature. So we came to the following conclusions:

  • 1. E. coli DH5α protein expression system will be affected by temperature.
  • 2. The change of temperature and the expression level of sfGFP are approximately linear.

    • We are very grateful to Fudan China for helping us in this part of the experiment. Other cooperation about us will be recorded on the Collaboration page, you can Click Here!

    After obtaining such a conclusion, it is important to eliminate the influence of temperature to the bacterial expression system. Therefore, as for the data processing, we mainly reflect the sfGFP intensity expressed by bacteria at a unit concentration by RFU. And the related RFU is used to reflect the change of expression intensity with different temperature in the experimental group compared with the control group. Their calculation methods are as follows: