Team:Shenzhen SFLS/Project

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<h1 class="hide">Project</h1>
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<p>This is our project, EcoPi. <br />
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<p>Eutrophication can be a very severe problem to the environment. In an effort to solve this problem, we want to build a genetic system which can test and remove the phosphate from water. In our idea, we want to make a product which can automatically detects phosphate and therefore remove it. And it must be a totally environmentally friendly one. And we began our designing.
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<img src="https://static.igem.org/mediawiki/2013hs/d/d9/Project_img_2.jpg" width="800" height="284" alt="" />  
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Frist we designed a genetic circuit which can test how the work has been done. We wanted to find a promoter which can only be induced by Pi starvation. Then there is a suppressor protein and an <span>rfp</span>. A double terminator is at the end of this device. This device can only work when the concentration of Pi is low. And the water will show red fluorescence. The red can also be a signal of ‘STOP’, which means the work has already finished.<br />
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Next we designed a second device for removing the phosphate. The promoter of this device is repressed by the protein in Device one, which means when the concentration of Pi is low, Device two will be limited and stop working. Then we wanted to find a CDS which can remove phosphate from water. And a <span>gfp</span>+LVA(degradation tag) with a terminator at the end. When this device is working, The CDS will remove the phosphate from water and meanwhile the gfp+LVA will show green fluorescence, just like the green traffic signal. This device is mainly used for removing phosphate.
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<p>This photo can show you how the two device are linked
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When we put our product into the water which has a very high concentration of phosphate, Device one will be limited and Device two will be activated. Then the water will show green fluorescence which means our product is removing Pi.
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After a period of time, due to Device two’s work, the concentration of Pi decreases, then Device one will be activated because of Pi starvation. The suppressor protein will repress Device two. Device one will work and show red fluorescence to tell us that the work has been finished.
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<p>That’s the idea of our project, we then started searching for files that support our idea. First we searched for a CDS which can digest phosphate. Then after weeks of searching we found Polyphosphate Kinase, also known as PPK. It is a kind of enzyme widely distributed in ecosystem, more importantly, it can be found in E-coli. Here this picture below can show you how it works.
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<p>We found its genetic sequence and we used PCR to made it a standard biobrick. Then we started to find the promoters of our product. Finally we found the Pi Starvation Promoter(BBa_K737023), it’s not on the kits so we again designed its primer and used PCR.</p>
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<p>For the protein we used TetR protein(BBa_C0040)</p>
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<p>Now it’s time for our final genetic circuit. Take a look!
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<p>Result</p>
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<p>After we finished our project, we started to test it. We test two part of our project. The first one is our Device One. We tested its response to Pi starvation. Here is the results diagram.</p>
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<p>In this diagram we can find that Device one can be induced by phosphate starvation. When the concentration of dipotassium phosphate is 0.03μM we can see that our sensor reached a peak because our promoter can be activated by Pi starvation.</p>
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<p>Next is our whole Device. We tested when the green fluorescence would vanish and red fluorescence occur. Here is another diagram.</p>
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<p>This diagram shows that after several hours the amount of green fluorescence decreases, which means the work has been done and red fluorescence will occur.</p>
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<p>This result clearly pointed out that our product can work smoothly. The Pi starvation promoter is working well too. Our experiments have reached a success at last.</p
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Latest revision as of 02:35, 22 June 2013

Project

This is our project, EcoPi.

In this page, you can see how it was designed and made.

Eutrophication can be a very severe problem to the environment. In an effort to solve this problem, we want to build a genetic system which can test and remove the phosphate from water. In our idea, we want to make a product which can automatically detects phosphate and therefore remove it. And it must be a totally environmentally friendly one. And we began our designing. Frist we designed a genetic circuit which can test how the work has been done. We wanted to find a promoter which can only be induced by Pi starvation. Then there is a suppressor protein and an rfp. A double terminator is at the end of this device. This device can only work when the concentration of Pi is low. And the water will show red fluorescence. The red can also be a signal of ‘STOP’, which means the work has already finished.
Next we designed a second device for removing the phosphate. The promoter of this device is repressed by the protein in Device one, which means when the concentration of Pi is low, Device two will be limited and stop working. Then we wanted to find a CDS which can remove phosphate from water. And a gfp+LVA(degradation tag) with a terminator at the end. When this device is working, The CDS will remove the phosphate from water and meanwhile the gfp+LVA will show green fluorescence, just like the green traffic signal. This device is mainly used for removing phosphate.

This photo can show you how the two device are linked When we put our product into the water which has a very high concentration of phosphate, Device one will be limited and Device two will be activated. Then the water will show green fluorescence which means our product is removing Pi.

After a period of time, due to Device two’s work, the concentration of Pi decreases, then Device one will be activated because of Pi starvation. The suppressor protein will repress Device two. Device one will work and show red fluorescence to tell us that the work has been finished.

That’s the idea of our project, we then started searching for files that support our idea. First we searched for a CDS which can digest phosphate. Then after weeks of searching we found Polyphosphate Kinase, also known as PPK. It is a kind of enzyme widely distributed in ecosystem, more importantly, it can be found in E-coli. Here this picture below can show you how it works.

We found its genetic sequence and we used PCR to made it a standard biobrick. Then we started to find the promoters of our product. Finally we found the Pi Starvation Promoter(BBa_K737023), it’s not on the kits so we again designed its primer and used PCR.

For the protein we used TetR protein(BBa_C0040)

Now it’s time for our final genetic circuit. Take a look!

Result

After we finished our project, we started to test it. We test two part of our project. The first one is our Device One. We tested its response to Pi starvation. Here is the results diagram.

In this diagram we can find that Device one can be induced by phosphate starvation. When the concentration of dipotassium phosphate is 0.03μM we can see that our sensor reached a peak because our promoter can be activated by Pi starvation.

Next is our whole Device. We tested when the green fluorescence would vanish and red fluorescence occur. Here is another diagram.

This diagram shows that after several hours the amount of green fluorescence decreases, which means the work has been done and red fluorescence will occur.

This result clearly pointed out that our product can work smoothly. The Pi starvation promoter is working well too. Our experiments have reached a success at last.

Retrieved from "http://2013hs.igem.org/Team:Shenzhen_SFLS/Project"