Difference between revisions of "Team:HBUT-China/Demonstrate"

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              <h1>Demonstrate</h1>
 
              <h1>Demonstrate</h1>
 
            </div>
 
            </div>
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          <p>We analyzed the experimental data through modeling.</p>
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          <small class="bigtitle">Response to nickel ions：</small>
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<small class="bigtitle">1.Assumptions:</small>
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<p>We added nickel ions in the medium of strain K2304002. After 2h (200rpm, 37 ℃, LB), we centrifuged the culture medium at 12000 r / min. The red precipitate appeared at the bottom of the centrifuge tube，while the blank control group produced very little, or none of the red precipitation. This indicates that KL2304002 can be induced by Ni ions to produce RFP.</p>
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<p>We made four assumptions in order to simplify some of the aspects of the model. Many similar assumptions have been made in the literature.</p>
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<p>The degradation of and other substances are linear.</p>
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<p>Each cell has the same state.</p>
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<p>The reaction in the process satisfies the first-order kinetic reaction.</p>
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<small class="bigtitle">2. Transcription Repression and Release Repression</small>
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<div align="center" style="margin:10px 0;">
 
<div align="center" style="margin:10px 0;">
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<img src="https://static.igem.org/mediawiki/2017/5/5f/Hbut-demon-1.png" alt="this is a photo">
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    <img src="//2017.igem.org/wiki/images/8/89/Hbut-newresult-1.png" alt="this is a photo">
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</div>
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    </div>
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<div align="center" style="margin:10px 0;">
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<small class="bigtitle">Specific response:</small>
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<img src="https://static.igem.org/mediawiki/2017/f/fd/Hbut-demon-2.png" alt="this is a photo">
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<p>In order to detect whether strain K2304002 is also induced by other ions,</p>
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</div>
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<p>We mixed samples using Cd<sup>2 +</sup>, Co<sup>2 +</sup>, Cu<sup>2 +</sup>, Fe<sup>3 +</sup>, and Ni<sup>2 +</sup>in LB medium, with a concentration of 0.01 mmol / L .</p>
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<div align="center" style="margin:10px 0;">
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<p>We cultured the samples at 200rpm, 37 ℃ overnight.</p>
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<img src="https://static.igem.org/mediawiki/2017/d/db/Hbut-demon-3.png" alt="this is a photo">
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<p>The next day we observed them with a fluorescence microscope. Excitation light is Blue background light.</p>
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</div>
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<div align="center" style="margin:10px 0;">
 
<div align="center" style="margin:10px 0;">
−
<img src="https://static.igem.org/mediawiki/2017/a/a7/Hbut-demon-4.png" alt="this is a photo">
+
    <img src="//2017.igem.org/wiki/images/4/4f/Hbut-newresult-2.png" alt="this is a photo">
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</div>
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    </div>
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<p>Using the assumption about the first-order kinetic reaction,</p>
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<p>Clearly, only the sample with Ni ions showed red fluorescence, the other groups had no red fluorescence. Note that the iron ions reacted with the substance in the culture medium，so that there is an impurity in the field of view.</p>
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<p>Give：</p>
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−
<div align="center" style="margin:10px 0;">
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<img src="https://static.igem.org/mediawiki/2017/b/b3/Hbut-demon-5.png" alt="this is a photo">
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−
</div>
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−
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<small class="bigtitle">3. Quasi-steady-state Approximation (QSSA)</small>
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<p>Ordinary differential equations are too difficult to be solved, in order to get the solution of the equations, quasi-steady-state approximation is necessary.</p>
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<p>We use the approximate conditions as little as possible and try to make the quasi-steady-state of matter consistent with the actual situation.</p>
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−
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−
<div align="center" style="margin:10px 0;">
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−
<img src="https://static.igem.org/mediawiki/2017/4/4a/Hbut-demon-6.png" alt="this is a photo">
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</div>
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<p>According to the formula’s deformation and derivation, </p>
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<p>it gives：</p>
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<div align="center" style="margin:10px 0;">
 
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<img src="https://static.igem.org/mediawiki/2017/5/5c/Hbut-demon-7.png" alt="this is a photo">
 
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</div>
 
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<small class="bigtitle">4. Incomplete Repression</small>
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<small class="bigtitle">Qualitative testing：</small>
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<p>Under incomplete repression, the bacteria express red fluorescent proteins without the presence of nickel ions.</p>
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<p>K304002 was cultured at different concentrations of nickel ions, and the fluorescence intensity was measured every hour. We found that different concentrations of nickel ions caused inconclusive differences in fluorescence intensity.</p>
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<p>The incomplete repression of promoters is a major issue in our fluorescence system. This issue was particularly observed in following diagram.</p>
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<p>The common formulation of the Hill equation is as follows:</p>
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<div align="center" style="margin:10px 0;">
 
<div align="center" style="margin:10px 0;">
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<img src="https://static.igem.org/mediawiki/2017/3/30/Hbut-demon-8.png" alt="this is a photo">
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    <img src="//2017.igem.org/wiki/images/1/1d/Hbut-newresult-3.png" alt="this is a photo">
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</div>
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    </div>
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<p>Consequently, we modeled incomplete repression by using the ratio of occupied promoter concentration to total promoter concentration.</p>
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<div align="center" style="margin:10px 0;">
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<img src="https://static.igem.org/mediawiki/2017/a/a9/Hbut-demon-9.png" alt="this is a photo">
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</div>
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<p>In view of the fact that is difficult to be measured and have same trend to , we used the concentration of nickel ions to approximate the equation.</p>
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<p>Therefore, we tried to find a way to perform qualitative testing. We used different concentrations of nickel ions to induce the strain in OD0.8, and detected the fluorescence intensity every other hour by fluorescence spectrophotometer. And finally we found with a nickel ion concentration of 10-6mmol / L to 10-4mmol / L, that the correlation between fluorescence intensity and nickel ion concentration is the highest. So it has the application value of qualitative detection.</p>
 
<div align="center" style="margin:10px 0;">
 
<div align="center" style="margin:10px 0;">
−
  <img src="https://static.igem.org/mediawiki/2017/9/95/Hbut-demon-10.png" alt="this is a photo">
+
    <img src="//2017.igem.org/wiki/images/2/29/Hbut-demon-new2.png" alt="this is a photo">
 
     </div>
 
     </div>
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<p>So, the fluorescence intensity can be obtained without considering the cytotoxicity of Nickel Ions:</p>
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<div align="center" style="margin:10px 0;">
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<p>This is the conclusion we have made so far, and if we continue to experiment, we believe we can find a better application mode, or an exact detectable interval. After that, we will be able to further study its measurement accuracy.</p>
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<img src="https://static.igem.org/mediawiki/2017/9/9d/Hbut-demon-11.png" alt="this is a photo">
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−
</div>
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<p>The para meters for the production function are:</p>
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<p><i>A</i>-basal expression level of promoter</p>
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<small class="bigtitle">Application：</small>
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<p><i>B</i>-maximal expression level of promoter</p>
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<p><i>K<sub>d</sub></i>-half maximal effective concentration of N-Ni</p>
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<p><i>n</i>-Hill coefficient for induction. </p>
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<p>Added Variable, The differential equation of was updated by:</p>
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<div align="center" style="margin:10px 0;">
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<img src="https://static.igem.org/mediawiki/2017/3/3b/Hbut-demon-12.png" alt="this is a photo">
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</div>
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<p>The solution of this differential equation is as follows：</p>
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<div align="center" style="margin:10px 0;">
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<img src="https://static.igem.org/mediawiki/2017/2/22/Hbut-demon-13.png" alt="this is a photo">
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</div>
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<small class="bigtitle">5.Application </small>
+
 
<p>We designed a kit, including 10ml centrifuge tubes, filter columns (containing 0.22 M filter membrane), the national standard concentration of nickel ion solution 50ml(3μg/L), pure water 20ml, engineering bacteria 5ml.</p>
 
<p>We designed a kit, including 10ml centrifuge tubes, filter columns (containing 0.22 M filter membrane), the national standard concentration of nickel ion solution 50ml(3μg/L), pure water 20ml, engineering bacteria 5ml.</p>
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<p>Instructions：
 
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<p>1. Add 3ml chloramphenicol containing LB medium and 30μL of original bacteria to the test tube. Incubate at 37°C 200rpm for overnight, and dilute the cultures to a target OD600 of 0.8;</p>
 
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<p>2.Take out 10ml centrifuge tubes for each sample besides one for blank one for stdrand. Add 2.7ml chloramphenicol containing LB medium and 30μL overnignt bacteria into each tube;</p>
 
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<p>3.Add 2.7ml chloramphenicol containing LB medium and 30μL of the overnight culture solution into each tube.</p>
 
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<p>4.Place the filter columns into each centrifuge tube.  Add 300μL pure water into one column，add 300μL nickel ion standard solution into another column, and add 300μL sample solution into the other columns.</p>
 
  
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<p>5. Detect the fluorescence after incubating at 37°C, 200rpm for 4hr.</p>
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<p>Here is the Instructions：</p>
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<div align="center" style="margin:10px 0;">
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<div align="center" style="margin:10px 0;">
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<img src="//2017.igem.org/wiki/images/3/3d/Hbut-demo-final.png" alt="this is a photo">
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    <img src="//2017.igem.org/wiki/images/2/29/Hbut-demon-new1.png" alt="this is a photo">
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</div>
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    </div>
 
        </div>
 
        </div>
 
         <div class="col-lg-2"></div>
 
         <div class="col-lg-2"></div>

Revision as of 01:47, 2 November 2017

    • \

Demonstrate

Response to nickel ions：

We added nickel ions in the medium of strain K2304002. After 2h (200rpm, 37 ℃, LB), we centrifuged the culture medium at 12000 r / min. The red precipitate appeared at the bottom of the centrifuge tube，while the blank control group produced very little, or none of the red precipitation. This indicates that KL2304002 can be induced by Ni ions to produce RFP.

this is a photo
Specific response:

In order to detect whether strain K2304002 is also induced by other ions,

We mixed samples using Cd2 +, Co2 +, Cu2 +, Fe3 +, and Ni2 +in LB medium, with a concentration of 0.01 mmol / L .

We cultured the samples at 200rpm, 37 ℃ overnight.

The next day we observed them with a fluorescence microscope. Excitation light is Blue background light.

this is a photo

Clearly, only the sample with Ni ions showed red fluorescence, the other groups had no red fluorescence. Note that the iron ions reacted with the substance in the culture medium，so that there is an impurity in the field of view.

Qualitative testing：

K304002 was cultured at different concentrations of nickel ions, and the fluorescence intensity was measured every hour. We found that different concentrations of nickel ions caused inconclusive differences in fluorescence intensity.

this is a photo

Therefore, we tried to find a way to perform qualitative testing. We used different concentrations of nickel ions to induce the strain in OD0.8, and detected the fluorescence intensity every other hour by fluorescence spectrophotometer. And finally we found with a nickel ion concentration of 10-6mmol / L to 10-4mmol / L, that the correlation between fluorescence intensity and nickel ion concentration is the highest. So it has the application value of qualitative detection.

this is a photo

This is the conclusion we have made so far, and if we continue to experiment, we believe we can find a better application mode, or an exact detectable interval. After that, we will be able to further study its measurement accuracy.

Application：

We designed a kit, including 10ml centrifuge tubes, filter columns (containing 0.22 M filter membrane), the national standard concentration of nickel ion solution 50ml(3μg/L), pure water 20ml, engineering bacteria 5ml.

Here is the Instructions：

this is a photo