Team:Tsinghua/Dataresult

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   <p>In this module, we hope we can apply LuxI protein to  produce AHL which can keep the signal transmitting.<br />
   <p>In this module, we hope we can apply LuxI protein to  produce AHL which can keep the signal transmitting.<br />
     Construct 1: <em>lux</em>I on pet15b</p>
     Construct 1: <em>lux</em>I on pet15b</p>
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   <p><img src="KI/untitled.jpg" width="273" height="163" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/a/ae/Untitled_TS.jpg" width="273" height="163" /></p>
   <p>To make sure the protein<em> Lux</em>I was produced we carried  out a SDS-page after purification using a Ni-column (LuxI was with a His tag).</p>
   <p>To make sure the protein<em> Lux</em>I was produced we carried  out a SDS-page after purification using a Ni-column (LuxI was with a His tag).</p>
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   <p><img src="KI/untitled2.jpg" width="554" height="287" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/4/4f/Untitled2_TS.jpg" width="554" height="287" /></p>
   <p class="comment">Figure0. A SDS-page showing the purification of LuxI.<br />
   <p class="comment">Figure0. A SDS-page showing the purification of LuxI.<br />
   The production of AHL needs LuxI protein as an enzyme  and a substrate molecule which is supposed to exist originally in E.<em>coli</em></p>
   The production of AHL needs LuxI protein as an enzyme  and a substrate molecule which is supposed to exist originally in E.<em>coli</em></p>
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     Our first construct consists of Lux Promoter which can  be activated by AHL and RFP.<br />
     Our first construct consists of Lux Promoter which can  be activated by AHL and RFP.<br />
     Construct 2: Plac-luxr-Plux-RFP on pSB1A2</p>
     Construct 2: Plac-luxr-Plux-RFP on pSB1A2</p>
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   <p><img src="KI/untitled3.jpg" width="554" height="221" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/0/06/Untitled3_TS.jpg" width="554" height="221" /></p>
   <p><em>the part 1-9H in the  igem 2012 kit was used</em></p>
   <p><em>the part 1-9H in the  igem 2012 kit was used</em></p>
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   <p><img src="KI/untitled4.jpg" alt="" width="492" height="294" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/2/21/Untitled4_TS.jpg" alt="" width="492" height="294" /></p>
   <p class="comment">Figure 1. A PCR result to confirm the accomplishment  of construct 1.</p>
   <p class="comment">Figure 1. A PCR result to confirm the accomplishment  of construct 1.</p>
   <p>Then we did a few tests to see if this construct could  successfully sense the signal of AHL and produce RFP as an indicator. To test  the efficiency of this AHL trigger, we directly added AHL into the E.<em>coli</em> and the photo below is taken 12h  after adding AHL.</p>
   <p>Then we did a few tests to see if this construct could  successfully sense the signal of AHL and produce RFP as an indicator. To test  the efficiency of this AHL trigger, we directly added AHL into the E.<em>coli</em> and the photo below is taken 12h  after adding AHL.</p>
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   <p><img src="KI/untitled5.jpg" width="478" height="317" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/c/c1/Untitled5_TS.jpg" width="478" height="317" /></p>
   <p class="comment">Figure2. Tests of AHL trigger. Different AHL molecules  and different concentration were set as the photo shows.</p>
   <p class="comment">Figure2. Tests of AHL trigger. Different AHL molecules  and different concentration were set as the photo shows.</p>
   <p>Next, we use fluorescence microscope to visualize the appearance of  RFP as the time goes. </p>
   <p>Next, we use fluorescence microscope to visualize the appearance of  RFP as the time goes. </p>
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   <p><img src="KI/untitled6.jpg" width="326" height="326" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/e/e3/Untitled6_TS.jpg" width="326" height="326" /></p>
   <p class="comment">Figure3. Without AHL, the bacteria didn&rsquo;t tend to  produce much RFP.</p>
   <p class="comment">Figure3. Without AHL, the bacteria didn&rsquo;t tend to  produce much RFP.</p>
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   <p><img src="KI/tsinghua_data1.jpg" width="206" height="281" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/9/9e/Tsinghua_data1.jpg" width="206" height="281" /></p>
   <p class="comment">Figure4. 1h after AHL was added (bright and dark).</p>
   <p class="comment">Figure4. 1h after AHL was added (bright and dark).</p>
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   <p><img src="KI/tsinghua_data2.jpg" width="206" height="281" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/f/f7/Tsinghua_data2.jpg" width="206" height="281" /></p>
   <p class="comment">Figure5. 2h after AHL was added.</p>
   <p class="comment">Figure5. 2h after AHL was added.</p>
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   <p><img src="KI/tsinghua_data3.jpg" width="268" height="268" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/c/c0/Tsinghua_data3.jpg" width="268" height="268" /></p>
   <p class="comment">Figure6. 3h after AHL was added.</p>
   <p class="comment">Figure6. 3h after AHL was added.</p>
   <p>After all these tests, we are convinced that this  construct does function as both a signal sensor and a signal releaser.</p>
   <p>After all these tests, we are convinced that this  construct does function as both a signal sensor and a signal releaser.</p>
   <p>After confirming construct2, we naturally want to  combine module I and II to see what will happen.<br />
   <p>After confirming construct2, we naturally want to  combine module I and II to see what will happen.<br />
     At the first, we just mix up these two kinds of  bacteria(one can produce AHL, the other sense it), the result was as  below(negative control was not red which isn&rsquo;t shown here).</p>
     At the first, we just mix up these two kinds of  bacteria(one can produce AHL, the other sense it), the result was as  below(negative control was not red which isn&rsquo;t shown here).</p>
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   <p><img src="KI/tsinghua_data4.jpg" width="387" height="290" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/d/d5/Tsinghua_data4.jpg" width="387" height="290" /></p>
   <p>Then, we decide to separately locate the two kinds of  bac and see if it could appear red in the contacting area.</p>
   <p>Then, we decide to separately locate the two kinds of  bac and see if it could appear red in the contacting area.</p>
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   <p><img src="KI/tsinghua_data5.jpg" alt="" width="388" height="291" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/4/45/Tsinghua_data5.jpg" alt="" width="388" height="291" /></p>
   <p>After all these tests, we decided to move on, so we  designed the next construct.<br />
   <p>After all these tests, we decided to move on, so we  designed the next construct.<br />
   Construct 3: Plac-luxr-Plux-RFP-Plac-LuxI on pet15b</p>
   Construct 3: Plac-luxr-Plux-RFP-Plac-LuxI on pet15b</p>
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   <p><img src="KI/dataTsinghua6.png" alt="" width="306" height="120" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/f/fe/DataTsinghua6.png" alt="" width="306" height="120" /></p>
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   <p><img src="KI/dataTsinghua7.png" width="178" height="161" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/2/24/DataTsinghua7.png" width="178" height="161" /></p>
   <p class="comment">Figure8. A enzyme digestion test showing that the  sample on lane 4 is positive.<br />
   <p class="comment">Figure8. A enzyme digestion test showing that the  sample on lane 4 is positive.<br />
   However, we didn&rsquo;t see any sign of AHL signaling  through this construct and we believe the arrangement of promoters is somehow  inappropriate. So we redesigned the plasmid and built construct 5.</p>
   However, we didn&rsquo;t see any sign of AHL signaling  through this construct and we believe the arrangement of promoters is somehow  inappropriate. So we redesigned the plasmid and built construct 5.</p>
   <p>Construct 5:Plac-luxr-Plux-RFP-LuxI on pet15b</p>
   <p>Construct 5:Plac-luxr-Plux-RFP-LuxI on pet15b</p>
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   <p><img src="KI/tsinghua_data8.jpg" width="317" height="169" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/7/7a/Tsinghua_data8.jpg" width="317" height="169" /></p>
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   <p><img src="KI/tsinghua_data9.jpg" alt="" width="251" height="151" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/c/cd/Tsinghua_data9.jpg" alt="" width="251" height="151" /></p>
   <p class="comment">Figure9. A PCR screen showing that lane 5 is positive.</p>
   <p class="comment">Figure9. A PCR screen showing that lane 5 is positive.</p>
   <p>Then we made dishes with AHL in the middle of the  plate only and spread E.<em>coli</em> with construct  5 on the whole plate hoping we can see the red firstly appear in the middle and  then spread out to the whole plate. </p>
   <p>Then we made dishes with AHL in the middle of the  plate only and spread E.<em>coli</em> with construct  5 on the whole plate hoping we can see the red firstly appear in the middle and  then spread out to the whole plate. </p>
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   <p><img src="KI/tsinghua_data10.jpg" width="417" height="313" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/8/83/Tsinghua_data10.jpg" width="417" height="313" /></p>
   <p class="comment">Figure10. AHL in the middle. We can see that only the  central part of the plate turned red.</p>
   <p class="comment">Figure10. AHL in the middle. We can see that only the  central part of the plate turned red.</p>
   <p>And we have known that concentration of AHL needs to  be higher than some certain value to activate the expression. So we also tried  to add a low level of AHL all over the plate and still with a high level of AHL  in the middle of the plate.</p>
   <p>And we have known that concentration of AHL needs to  be higher than some certain value to activate the expression. So we also tried  to add a low level of AHL all over the plate and still with a high level of AHL  in the middle of the plate.</p>
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   <p><img src="KI/tsinghua_data11.jpg" alt="" width="492" height="547" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/4/4b/Tsinghua_data11.jpg" alt="" width="492" height="547" /></p>
   <p class="comment">Figure11. a, E.<em>coli</em> with construct 2, a red patch appears where there is AHL(yet the photo is not  clear); b-f, 200μl water with 0, 0.05, 0.2,  0.5, 2μl AHL(10-4mol/L). RFP appear all  over the plate in c-f, while some on the edge in b.</p>
   <p class="comment">Figure11. a, E.<em>coli</em> with construct 2, a red patch appears where there is AHL(yet the photo is not  clear); b-f, 200μl water with 0, 0.05, 0.2,  0.5, 2μl AHL(10-4mol/L). RFP appear all  over the plate in c-f, while some on the edge in b.</p>
   <p>The results were not perfect and not able to prove the  successful transmission of AHL. However, the result show us some hope that the  system will work as long as the circumstances are appropriately modified. </p>
   <p>The results were not perfect and not able to prove the  successful transmission of AHL. However, the result show us some hope that the  system will work as long as the circumstances are appropriately modified. </p>
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   <p>The work done before was not just for seeing some RFP,  we build this system to see if we can get some amplified signal transmitting  through some distance that can be processed in a signal center. Then we design  construct 6&amp;7.</p>
   <p>The work done before was not just for seeing some RFP,  we build this system to see if we can get some amplified signal transmitting  through some distance that can be processed in a signal center. Then we design  construct 6&amp;7.</p>
   <p>Construct6: <span style=" color:#F06">Plux</span>-<span style="color:#0CF">LacI</span>-<span style="color:#0CF">LuxI</span>-<span style=" color:#F06">Plac</span>-<span style="color:#0CF">LuxR</span>-<span style="color:#0CF">LasR</span>-<span style=" color:#F06">Plac</span>-<span>LacO</span>-<span style="color:#0CF">cI</span>-<span style=" color:#F06">Plas</span>-<span style="color:#0CF">LasI</span>-<span style=" color:#F06">Plas/cI</span>-<span style="color:#0CF">RFP</span></p>
   <p>Construct6: <span style=" color:#F06">Plux</span>-<span style="color:#0CF">LacI</span>-<span style="color:#0CF">LuxI</span>-<span style=" color:#F06">Plac</span>-<span style="color:#0CF">LuxR</span>-<span style="color:#0CF">LasR</span>-<span style=" color:#F06">Plac</span>-<span>LacO</span>-<span style="color:#0CF">cI</span>-<span style=" color:#F06">Plas</span>-<span style="color:#0CF">LasI</span>-<span style=" color:#F06">Plas/cI</span>-<span style="color:#0CF">RFP</span></p>
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   <p><img src="KI/tsinghua_data12.jpg" width="554" height="154" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/8/8c/Tsinghua_data12.jpg" width="554" height="154" /></p>
   <p>The advanced version of our design consists of too  many parts which can hardly assembled one by one. So we decided to use overlap  PCR to get the construct. However, we have only finished part of the plan and  are concentrating on the rest.</p>
   <p>The advanced version of our design consists of too  many parts which can hardly assembled one by one. So we decided to use overlap  PCR to get the construct. However, we have only finished part of the plan and  are concentrating on the rest.</p>
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   <p><img src="KI/tsinghua_data13.jpg" width="271" height="205" /><img src="KI/tsinghua_data14.jpg" width="247" height="205" /><img src="KI/tsinghua_data15.jpg" alt="" width="202" height="205" /></p>
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   <p><img src="https://static.igem.org/mediawiki/2012/4/49/Tsinghua_data13.jpg" width="271" height="205" /><img src="https://static.igem.org/mediawiki/2012/9/96/Tsinghua_data14.jpg" width="247" height="205" /><img src="https://static.igem.org/mediawiki/2012/5/5a/Tsinghua_data15.jpg" alt="" width="202" height="205" /></p>
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   <p align="center"><img src="KI/tsinghua_data16.jpg" alt="" width="215" height="212" /></p>
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   <p align="center"><img src="https://static.igem.org/mediawiki/2012/e/e4/Tsinghua_data16.jpg" alt="" width="215" height="212" /></p>
   <p class="comment" align="center">Figure12. Some results of a complex overlap PCR to get  construct 6.</p>
   <p class="comment" align="center">Figure12. Some results of a complex overlap PCR to get  construct 6.</p>
   <p>Construct7:  And-not  Gate</p><p>
   <p>Construct7:  And-not  Gate</p><p>
     And-not  gate works when one signal exists while the other one not. We designed this  part based on two kinds of AHL:C6HSL(signal A) and C12HSL(signal B). </p>
     And-not  gate works when one signal exists while the other one not. We designed this  part based on two kinds of AHL:C6HSL(signal A) and C12HSL(signal B). </p>
   <p>&nbsp;</p>
   <p>&nbsp;</p>
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<p align="center"><img src="KI/tsinghua_data17.jpg" alt="" width="553" height="184" /></p>
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/9/99/Tsinghua_data17.jpg" alt="" width="553" height="184" /></p>
<p>As the  picture above shows, C6HSL could activate the promoter Plux and the promoter  Plas is activated by C12HSL. In the design of this part, we also added cI  operator downstream of Plas. As a result, the promoter Plas could be activated  by signal B but repressed by cI protein. The expression of cI protein is under  the control of Plux, which could be activated by signal A. RFP is controled by  Hybrid promoter(Plas and cI operator), which would work as a reporter. </p>
<p>As the  picture above shows, C6HSL could activate the promoter Plux and the promoter  Plas is activated by C12HSL. In the design of this part, we also added cI  operator downstream of Plas. As a result, the promoter Plas could be activated  by signal B but repressed by cI protein. The expression of cI protein is under  the control of Plux, which could be activated by signal A. RFP is controled by  Hybrid promoter(Plas and cI operator), which would work as a reporter. </p>
<p>In a  word, the expression of RFP is only on the condition that A is absent and B is  present. </p>
<p>In a  word, the expression of RFP is only on the condition that A is absent and B is  present. </p>
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   <p>This design is simpler than the last one and we also chose  overlap PCR to construct this part. Fortunately, we finished this part and  obtained the effect we want. The pictures below show part of the construction process.</p>
   <p>This design is simpler than the last one and we also chose  overlap PCR to construct this part. Fortunately, we finished this part and  obtained the effect we want. The pictures below show part of the construction process.</p>
</p>
</p>
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<img src="KI/tsinghua_data18.jpg" alt="" width="548" height="409" /></div>
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<img src="https://static.igem.org/mediawiki/2012/6/62/Tsinghua_data18.jpg" alt="" width="548" height="409" /></div>
<p>Figure13. Results of overlap PCR to get construct 7.  The picture on the left shows the acquirement of two of the single parts  (Hybrid promotor (HP) and RFP). The Picture on the right shows that we obtained  the fusion product of HP and RFP. (F means Failure, while S means Success. the  fusion product is marked by red arrow)</p>
<p>Figure13. Results of overlap PCR to get construct 7.  The picture on the left shows the acquirement of two of the single parts  (Hybrid promotor (HP) and RFP). The Picture on the right shows that we obtained  the fusion product of HP and RFP. (F means Failure, while S means Success. the  fusion product is marked by red arrow)</p>
<p align="center">&nbsp;</p>
<p align="center">&nbsp;</p>

Latest revision as of 16:12, 25 September 2012

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Data & Result

Experiment and Data

In this part we will show the experiment we have designed and carried out along with the results. Only valid and positive results will be displayed.
Our project is based on three modules, so is the part of experiment. So we will introduce our work to you according to different modules.

Module I. Signal transmission

In this module, we hope we can apply LuxI protein to produce AHL which can keep the signal transmitting.
Construct 1: luxI on pet15b

To make sure the protein LuxI was produced we carried out a SDS-page after purification using a Ni-column (LuxI was with a His tag).

Figure0. A SDS-page showing the purification of LuxI.
The production of AHL needs LuxI protein as an enzyme and a substrate molecule which is supposed to exist originally in E.coli

Module II. Signal sensing and releasing

According to the project design, we are using quorum sensing system in gram negative bacteria as our signaling system.
Our first construct consists of Lux Promoter which can be activated by AHL and RFP.
Construct 2: Plac-luxr-Plux-RFP on pSB1A2

the part 1-9H in the igem 2012 kit was used

Figure 1. A PCR result to confirm the accomplishment of construct 1.

Then we did a few tests to see if this construct could successfully sense the signal of AHL and produce RFP as an indicator. To test the efficiency of this AHL trigger, we directly added AHL into the E.coli and the photo below is taken 12h after adding AHL.

Figure2. Tests of AHL trigger. Different AHL molecules and different concentration were set as the photo shows.

Next, we use fluorescence microscope to visualize the appearance of RFP as the time goes.

Figure3. Without AHL, the bacteria didn’t tend to produce much RFP.

Figure4. 1h after AHL was added (bright and dark).

Figure5. 2h after AHL was added.

Figure6. 3h after AHL was added.

After all these tests, we are convinced that this construct does function as both a signal sensor and a signal releaser.

After confirming construct2, we naturally want to combine module I and II to see what will happen.
At the first, we just mix up these two kinds of bacteria(one can produce AHL, the other sense it), the result was as below(negative control was not red which isn’t shown here).

Then, we decide to separately locate the two kinds of bac and see if it could appear red in the contacting area.

After all these tests, we decided to move on, so we designed the next construct.
Construct 3: Plac-luxr-Plux-RFP-Plac-LuxI on pet15b

Figure8. A enzyme digestion test showing that the sample on lane 4 is positive.
However, we didn’t see any sign of AHL signaling through this construct and we believe the arrangement of promoters is somehow inappropriate. So we redesigned the plasmid and built construct 5.

Construct 5:Plac-luxr-Plux-RFP-LuxI on pet15b

Figure9. A PCR screen showing that lane 5 is positive.

Then we made dishes with AHL in the middle of the plate only and spread E.coli with construct 5 on the whole plate hoping we can see the red firstly appear in the middle and then spread out to the whole plate.

Figure10. AHL in the middle. We can see that only the central part of the plate turned red.

And we have known that concentration of AHL needs to be higher than some certain value to activate the expression. So we also tried to add a low level of AHL all over the plate and still with a high level of AHL in the middle of the plate.

Figure11. a, E.coli with construct 2, a red patch appears where there is AHL(yet the photo is not clear); b-f, 200μl water with 0, 0.05, 0.2, 0.5, 2μl AHL(10-4mol/L). RFP appear all over the plate in c-f, while some on the edge in b.

The results were not perfect and not able to prove the successful transmission of AHL. However, the result show us some hope that the system will work as long as the circumstances are appropriately modified.

Module III. Signal Center

The work done before was not just for seeing some RFP, we build this system to see if we can get some amplified signal transmitting through some distance that can be processed in a signal center. Then we design construct 6&7.

Construct6: Plux-LacI-LuxI-Plac-LuxR-LasR-Plac-LacO-cI-Plas-LasI-Plas/cI-RFP

The advanced version of our design consists of too many parts which can hardly assembled one by one. So we decided to use overlap PCR to get the construct. However, we have only finished part of the plan and are concentrating on the rest.

Figure12. Some results of a complex overlap PCR to get construct 6.

Construct7:  And-not Gate

And-not gate works when one signal exists while the other one not. We designed this part based on two kinds of AHL:C6HSL(signal A) and C12HSL(signal B).

 

As the picture above shows, C6HSL could activate the promoter Plux and the promoter Plas is activated by C12HSL. In the design of this part, we also added cI operator downstream of Plas. As a result, the promoter Plas could be activated by signal B but repressed by cI protein. The expression of cI protein is under the control of Plux, which could be activated by signal A. RFP is controled by Hybrid promoter(Plas and cI operator), which would work as a reporter.

In a word, the expression of RFP is only on the condition that A is absent and B is present.

This design is simpler than the last one and we also chose overlap PCR to construct this part. Fortunately, we finished this part and obtained the effect we want. The pictures below show part of the construction process.

Figure13. Results of overlap PCR to get construct 7. The picture on the left shows the acquirement of two of the single parts (Hybrid promotor (HP) and RFP). The Picture on the right shows that we obtained the fusion product of HP and RFP. (F means Failure, while S means Success. the fusion product is marked by red arrow)