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

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{{HBUT-China}}
 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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        <li><img src="https://static.igem.org/mediawiki/2017/5/55/HBUT-logo2.png" height="45"/></li>
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        \
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<h1>Demonstrate</h1>
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      <div class="oneByOne_item">
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<h3>Gold Medal Criterion #4</h3>
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        <a href="javascript:;"><img src="https://static.igem.org/mediawiki/2017/5/55/HBUT-logo2.png" width="300" class="wp1_3 slide1_bot"/></a>                
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        <span class="txt1">Nickel ion!</span>      
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<p>
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        <span class="txt2">Heavy metal pollutants!</span>                                  
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve a their gold medals!
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        <span class="txt3 short">For this year¡¯s iGEM Competition, our team will be focusing on putting forward a biological approach to detect nickel ions as a heavy metal contaminant in the natural environment</span>                                   
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Please see the <a href="https://2017.igem.org/Judging/Medals">2017 Medals Page</a> for more information.
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        <span class="txt1 blue txt_right2">In order to achieve this goal, we use a nickel-sensitive repressive promoter (pncrA)</span>      
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</p>
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        <span class="txt1 blue txt_right2">In the absence of nickel ions, the repressor protein (NcrB) binds to the promoter, preventing the expression of the subsequent gene</span>
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            <div class="page-header">
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              <h1>Demonstrate</h1>
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            </div>
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          <small class="bigtitle">Response to nickel ions：</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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<div align="center" style="margin:10px 0;">
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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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<small class="bigtitle">Specific response:</small>
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<p>In order to detect whether strain K2304002 is also induced by other ions,</p>
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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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<p>We cultured the samples at 200rpm, 37 ℃ overnight.</p>
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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 align="center" style="margin:10px 0;">
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    <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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<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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<div class="column half_size">
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<small class="bigtitle">Qualitative testing：</small>
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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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<div align="center" style="margin:10px 0;">
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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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<h4> What should we do for our demonstration?</h4>
 
  
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<h5> Standard teams </h5>
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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>
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<div align="center" style="margin:10px 0;">
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    <img src="//2017.igem.org/wiki/images/f/f3/Hbut-demon-new2.png" alt="this is a photo">
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    </div>
  
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<p>  
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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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If you have built a proof of concept system, you can demonstrate it working under real world conditions. If you have built a biological device that is intended to be a sensor, can you show it detecting whatever it is intended to sense. If it is intended to work in the field, you can show how this might work using a simulated version in the lab, or a simulation of your device in the field.<strong> Please note biological materials must not be taken out of the lab</strong>.
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<h5> Special track teams </h5>
 
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Special track teams can achieve this medal criterion by bringing their work to the Jamboree and showcasing it in the track event. Art & Design, Measurement, Hardware and Software tracks will all have showcase events at the Giant Jamboree.<strong> Please note biological materials must not be taken out of the lab</strong>.
 
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</p>
 
  
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<small class="bigtitle">Application：</small>
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<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>Here is the Instructions：</p>
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<div align="center" style="margin:10px 0;">
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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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Latest revision as of 01:48, 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