Team:Tsinghua

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<li><a href="#A">Signal transmission</a></li>
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<li><a href="#B">Signal sensing and releasing</a></li>
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<li><a href="#C">Signal Center</a></li>
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  <h3><em>Experiment and Data</em></h3>
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  <p>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.<br />
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    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.</p>
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  <h4><a name = "A">Module I. Signal transmission</a></h4>
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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 />
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    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>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 class="comment">Figure0. A SDS-page showing the purification of LuxI.<br />
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  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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  <h4><a name="B">Module II. Signal sensing and releasing</a></h4>
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  <p>According to the project design, we are using quorum  sensing system in gram negative bacteria as our signaling system.<br />
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    Our first construct consists of Lux Promoter which can  be activated by AHL and RFP.<br />
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    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><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 class="comment">Figure 1. A PCR result to confirm the accomplishment  of construct 1.</p>
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  <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 class="comment">Figure2. Tests of AHL trigger. Different AHL molecules  and different concentration were set as the photo shows.</p>
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  <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 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 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 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 class="comment">Figure6. 3h after AHL was added.</p>
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  <p>After all these tests, we are convinced that this  construct does function as both a signal sensor and a signal releaser.</p>
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  <p>After confirming construct2, we naturally want to  combine module I and II to see what will happen.<br />
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    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>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>After all these tests, we decided to move on, so we  designed the next construct.<br />
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  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="KI/dataTsinghua7.png" width="178" height="161" /></p>
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  <p class="comment">Figure8. A enzyme digestion test showing that the  sample on lane 4 is positive.<br />
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  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>
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  <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="KI/tsinghua_data9.jpg" alt="" width="251" height="151" /></p>
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  <p class="comment">Figure9. A PCR screen showing that lane 5 is positive.</p>
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  <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 class="comment">Figure10. AHL in the middle. We can see that only the  central part of the plate turned red.</p>
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  <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 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>
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  <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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  <h4><a name="C">Module III. Signal Center</a></h4>
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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>
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  <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>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 align="center"><img src="KI/tsinghua_data16.jpg" alt="" width="215" height="212" /></p>
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  <p class="comment" align="center">Figure12. Some results of a complex overlap PCR to get  construct 6.</p>
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  <p>Construct7:  And-not  Gate</p><p>
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    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>
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  <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>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>
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<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>
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</p>
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<img src="KI/tsinghua_data18.jpg" alt="" width="548" height="409" /></div>
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<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>
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<p align="center">&nbsp;</p>
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Latest revision as of 16:41, 26 September 2012

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Tsinghua iGEM Team 2012

 

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