Team:Uppsala

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<h2>Bactissiles: The future of microbial combat</h2>
<h2>Bactissiles: The future of microbial combat</h2>
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<p>Destabilized ecosystems and disturbed gut floras are both consequences of treatments that lack selectivity. More efficient and precise methods are needed. This year we, the Uppsala iGEM team, tries to widen the view and find new possibilities with engineered bacteria. By developing a system that homes towards a target and secretes an affectant, we can ensure a specific outcome. Such a system could have applications in a number of different fields, though we have chosen to put this into practice in a pinpointing pathogen-killing approach. In our prototype system, introduced in<i> E. coli </i>, we hijack the quorum sensing system of the gut pathogen <i> Yersinia enterocolitica </i>. Our bacteria will be able to sense the presence of the pathogen, accumulate in its vicinity and emit a target-specific bacteriocin, leaving the remaining gut flora intact. The era of mass destruction is over. Welcome the missile bacteria, the Bactissile! .</p>
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<p>Destabilized ecosystems and disturbed gut floras are both consequences of treatments that lack selectivity. More efficient and precise methods are needed. This year we, the Uppsala iGEM team, try to widen the view and find new possibilities with engineered bacteria. By developing a system that homes towards a target and secretes an affectant, we can ensure a specific outcome. Such a system could have applications in a number of different fields, though we have chosen to put this into practice in a pinpointing pathogen-killing approach. In our prototype system, introduced in<i> E. coli </i>, we hijack the quorum sensing system of the gut pathogen <i> Yersinia enterocolitica </i>. Our bacteria will be able to sense the presence of the pathogen, accumulate in its vicinity and emit a target-specific bacteriocin, leaving the remaining gut flora intact. The era of mass destruction is over. Welcome the missile bacteria, the Bactissile!</p><br>
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<h2>Assembly Plan</h2>
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<img class="schedule" src="https://static.igem.org/mediawiki/2014/a/ac/Uppsala2014_Kopplingsschema_final.png"</img>
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<tr><td></td><td><h2>What we have created</h2></td></tr>
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<tr><td><img class="schedule" src="https://static.igem.org/mediawiki/2014/e/ea/Sum_up_Uppsala14.png"</img></td>
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<td><p>Our creation, the Bactissile, is a missile bacteria, containing a two-mode system. Initially, the Bactissile will be in the <b>Tracking mode</b>, in which it will be producing a lot of the mobility protein, CheZ and a silencing sRNA, which will silence the expression of the Bactissiles weapon, colicin Fy. In this state, the Bactissile will be taking big leaps, randomly searching for its target, <i>Y. enterocolitica</i>. <br><br>When <i>Y. enterocolitica</i> enters the vicinity of the Bactissile, the Bactissile will switch to its <b>Attack mode</b>. In this state, the Bactissile will stop the production of CheZ and the sRNA and initiate the production of its weapon, the bacteriocin colicin Fy. The Bactissile will start tumbling close to the target, emitting its weapon. When the concentration of colicin Fy reaches a threshold value, the dosage will be lethal for <i>Y. enterocolitica</i>. Colicin Fy is only harmful for <i>Y. enterocolitica</i>, which entails that the beneficial gut flora in the Bactissile's surroundings will remain unharmed.</p></td></tr></table>
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<h1>Main Result</h1>
<h1>Main Result</h1>
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<h2>The Sensing System</h2>
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<p><br><i>Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:<br>
<p><br><i>Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:<br>
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1. pSB3C17-B0032-yenbox_WT-GFP<br>
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1. pSB3C17-yenbox_WT-B0032-GFP<br>
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2. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR<br>
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2. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23101-B0034-YenR<br>
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3. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR<br>
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3. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23110-B0034-YenR<br>
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4. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR</i></p>
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4. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23102-B0034-YenR</i></p>
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<p>By constructing the measuremnt construct <a href="http://parts.igem.org/Part:BBa_K1381008">BBa_K1381008</a> (yenbox_WT-B0032-GFP) and performing double transformation together with one of the constructs producting the activator YenR <a href="http://parts.igem.org/Part:BBa_K1381005">BBa_K1381005</a> (J23110-B0034-YenR), <a href="http://parts.igem.org/Part:BBa_K1381006">BBa_K1381006</a> (J23102-B0034-YenR) and <a href="http://parts.igem.org/Part:BBa_K1381007">BBa_K1381007</a> (J23101-B0034-YenR). We managed to show that the activator YenR works perfectly fine in E. coli and that it recognise the recognition region, the yenbox and induces the strength of the promoter fused with it. By measuring the production of the green fluorescence protein GFP using a flow cytometer, we could see that we got a five-fold induction when YenR with the strongest promoter out of the three used were present.
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<p>The measurement construct yenbox_WT-B0032-GFP was transformed into strains containing expression cassettes producing the activator YenR. Using a flow cytometer, we managed to show that YenR was working perfectly fine in <i>E. coli</i> and that it recognised the luxbox homolog, the yenbox, and induced the expression level. With the strongest YenR producing expression cassette we succeeded in getting a five-fold increase of the green fluorescence protein GFP production.
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<a href="https://2014.igem.org/Team:Uppsala/Project_Sensing">Read more about the Sensing System</a>
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<h2>Sensing Result</h2>
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<h2 class="border_h2_right">The Targeting System</h2>
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<p><br><i>Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:<br>
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<img class="main_pic_left" src="https://static.igem.org/mediawiki/2014/9/91/CheZswarmassay_Uppsala14.PNG">
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1. pSB3C17-B0032-yenbox_WT-GFP<br>
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2. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR<br>
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3. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR<br>
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4. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR</i></p>
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<img class="main_pic_left" src="https://static.igem.org/mediawiki/2014/b/b3/YenSystem_Char_Uppsala14.png">
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<p style="margin-left: 20px;"><br><i>Figure 1. A, B and C show three different swarmplate assays. 1: Motile strain RP437 (positive control), 2: cheZ mutant (negative control), 3: J23100-B0034-cheZ, 4: J23113-B0034-cheZ, 5: J23114-B0034-cheZ</i></p>
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<p>By constructing the measuremnt construct <a href="http://parts.igem.org/Part:BBa_K1381008">BBa_K1381008</a> (yenbox_WT-B0032-GFP) and performing double transformation together with one of the constructs producting the activator YenR <a href="http://parts.igem.org/Part:BBa_K1381005">BBa_K1381005</a> (J23110-B0034-YenR), <a href="http://parts.igem.org/Part:BBa_K1381006">BBa_K1381006</a> (J23102-B0034-YenR) and <a href="http://parts.igem.org/Part:BBa_K1381007">BBa_K1381007</a> (J23101-B0034-YenR). We managed to show that the activator YenR works perfectly fine in E. coli and that it recognise the recognition region, the yenbox and induces the strength of the promoter fused with it. By measuring the production of the green fluorescence protein GFP using a flow cytometer, we could see that we got a five-fold induction when YenR with the strongest promoter out of the three used were present.
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<p>We managed to restore chemotaxis in non-motile mutant strains, by reintroducing the cheZ gene on plasmids into the cheZ-knockout <i>E. coli</i>. Three promoters of different strengths were tested in combination with our construct and were shown to induce different levels of motility.</p>
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<a href="https://2014.igem.org/Team:Uppsala/Project_Targeting">Read more about the Targeting System</a>  
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<h2>The Killing System</h2>
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<p><br><i>Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:<br>
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<p><br><i>Figure 2. The picture shows the result from an experiment to test the killing efficiency of colicin Fy. On the left Y. enterocolitica has been grown in liquid culture together with colicin Fy. The right plate is a negative control, where Y. enterocolitica has grown in the same conditions in liquid culture without any colicin Fy added.</p>
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1. pSB3C17-B0032-yenbox_WT-GFP<br>
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2. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR<br>
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3. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR<br>
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4. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR</i></p>
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<img class="main_pic_left" src="https://static.igem.org/mediawiki/2014/b/b3/YenSystem_Char_Uppsala14.png">
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<p>By constructing the measuremnt construct <a href="http://parts.igem.org/Part:BBa_K1381008">BBa_K1381008</a> (yenbox_WT-B0032-GFP) and performing double transformation together with one of the constructs producting the activator YenR <a href="http://parts.igem.org/Part:BBa_K1381005">BBa_K1381005</a> (J23110-B0034-YenR), <a href="http://parts.igem.org/Part:BBa_K1381006">BBa_K1381006</a> (J23102-B0034-YenR) and <a href="http://parts.igem.org/Part:BBa_K1381007">BBa_K1381007</a> (J23101-B0034-YenR). We managed to show that the activator YenR works perfectly fine in E. coli and that it recognise the recognition region, the yenbox and induces the strength of the promoter fused with it. By measuring the production of the green fluorescence protein GFP using a flow cytometer, we could see that we got a five-fold induction when YenR with the strongest promoter out of the three used were present.
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<p>We managed to produce the bacteriocin colicin Fy. By fusing it with a His-tag we could perform an SDS-page gel and prove its presence. To analyse the colicin Fy’s killing efficiency, we let <i>Y. enterocolita</i> grow with and without colicin Fy in liquid cultures and compared the result. Fig. 2 shows these two  cultures plated. On the left plate, the <i>Y. enterocolitica</i> had grown with the colicin Fy added, while the right plate is the negative control, where <i>Y. enterocolitica</i> had grown without any colicin Fy. If we compare the two plates, we can see that there is a clear difference in the amount of <i>Y. enterocolitica</i> colonies.
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<p>By constructing the measuremnt construct <a href="http://parts.igem.org/Part:BBa_K1381008">BBa_K1381008</a> (yenbox_WT-B0032-GFP) and performing double transformation together with one of the constructs producting the activator YenR <a href="http://parts.igem.org/Part:BBa_K1381005">BBa_K1381005</a> (J23110-B0034-YenR), <a href="http://parts.igem.org/Part:BBa_K1381006">BBa_K1381006</a> (J23102-B0034-YenR) and <a href="http://parts.igem.org/Part:BBa_K1381007">BBa_K1381007</a> (J23101-B0034-YenR). We managed to show that the activator YenR works perfectly fine in E. coli and that it recognise the recognition region, the yenbox and induces the strength of the promoter fused with it. By measuring the production of the green fluorescence protein GFP using a flow cytometer, we could see that we got a five-fold induction when YenR with the strongest promoter out of the three used were present.
 
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<p><br><i>Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:<br>
 
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1. pSB3C17-B0032-yenbox_WT-GFP<br>
 
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2. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR<br>
 
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3. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR<br>
 
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4. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR</i></p>
 
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<p>The perpose of the targeting system is to ensure that our Bactissile will be as close to the pathogen as possible upon producing the killing bacteriocin. We chose to achieve this by controlling the Bactissiles chemotaxis through the protein CheZ. The targeting module is connected to the sensing module, which induces a lower production of CheZ when the Bactissile is closer to the pathogen, causing the Bactissile to stop. This way, we hoped to be able to control chemotaxis.
 
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After successfully assembling a promoter, an rbs and cheZ, we did manage to restore chemotaxis in one test by inserting this construct in a cheZ knock-out. However, since this only happened in one isolated instance, this is not evidence enough to prove the success of our construct, however it does indicate that through more rigorous and consisted testing more reliable results could be obtained. We therefore consider to have proved that our system could work, although at this moment we don’t have any definitive proof.
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<a href="https://2014.igem.org/Team:Uppsala/Project_Killing">Read more about the Killing System</a>  
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<p>The goal of this years project is to kill the gut pathogen Yersinina enterocolitica. this will be done by using the bacteriocin colicin FY. The plan was to fuse the bacteriocin with an export-tag but due to failures in assembling in the end we had to do our characterization by lysating our cells and take the bacteriocine from that supernatant. Doing this we were able to make a SDS-page and show the bacteriocins existance and further on adding the lysate to living yersinia culures. Seen in the picture are 2 plates of Y.enterocolitacas, the left one grown in a liquid culture with bacteriocine added while the other one is a negative control. There is a clear diference in the amount of yersinias so we can then show that the colicin is a succsess.
 
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Latest revision as of 21:26, 17 October 2014

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  • Bactissiles - The future of microbial combat

Bactissiles: The future of microbial combat

Destabilized ecosystems and disturbed gut floras are both consequences of treatments that lack selectivity. More efficient and precise methods are needed. This year we, the Uppsala iGEM team, try to widen the view and find new possibilities with engineered bacteria. By developing a system that homes towards a target and secretes an affectant, we can ensure a specific outcome. Such a system could have applications in a number of different fields, though we have chosen to put this into practice in a pinpointing pathogen-killing approach. In our prototype system, introduced in E. coli , we hijack the quorum sensing system of the gut pathogen Yersinia enterocolitica . Our bacteria will be able to sense the presence of the pathogen, accumulate in its vicinity and emit a target-specific bacteriocin, leaving the remaining gut flora intact. The era of mass destruction is over. Welcome the missile bacteria, the Bactissile!


What we have created

Our creation, the Bactissile, is a missile bacteria, containing a two-mode system. Initially, the Bactissile will be in the Tracking mode, in which it will be producing a lot of the mobility protein, CheZ and a silencing sRNA, which will silence the expression of the Bactissiles weapon, colicin Fy. In this state, the Bactissile will be taking big leaps, randomly searching for its target, Y. enterocolitica.

When Y. enterocolitica enters the vicinity of the Bactissile, the Bactissile will switch to its Attack mode. In this state, the Bactissile will stop the production of CheZ and the sRNA and initiate the production of its weapon, the bacteriocin colicin Fy. The Bactissile will start tumbling close to the target, emitting its weapon. When the concentration of colicin Fy reaches a threshold value, the dosage will be lethal for Y. enterocolitica. Colicin Fy is only harmful for Y. enterocolitica, which entails that the beneficial gut flora in the Bactissile's surroundings will remain unharmed.


Main Result

The Sensing System


Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:
1. pSB3C17-yenbox_WT-B0032-GFP
2. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23101-B0034-YenR
3. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23110-B0034-YenR
4. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23102-B0034-YenR

The measurement construct yenbox_WT-B0032-GFP was transformed into strains containing expression cassettes producing the activator YenR. Using a flow cytometer, we managed to show that YenR was working perfectly fine in E. coli and that it recognised the luxbox homolog, the yenbox, and induced the expression level. With the strongest YenR producing expression cassette we succeeded in getting a five-fold increase of the green fluorescence protein GFP production.


Read more about the Sensing System

The Targeting System


Figure 1. A, B and C show three different swarmplate assays. 1: Motile strain RP437 (positive control), 2: cheZ mutant (negative control), 3: J23100-B0034-cheZ, 4: J23113-B0034-cheZ, 5: J23114-B0034-cheZ

We managed to restore chemotaxis in non-motile mutant strains, by reintroducing the cheZ gene on plasmids into the cheZ-knockout E. coli. Three promoters of different strengths were tested in combination with our construct and were shown to induce different levels of motility.


Read more about the Targeting System

The Killing System


Figure 2. The picture shows the result from an experiment to test the killing efficiency of colicin Fy. On the left Y. enterocolitica has been grown in liquid culture together with colicin Fy. The right plate is a negative control, where Y. enterocolitica has grown in the same conditions in liquid culture without any colicin Fy added.

We managed to produce the bacteriocin colicin Fy. By fusing it with a His-tag we could perform an SDS-page gel and prove its presence. To analyse the colicin Fy’s killing efficiency, we let Y. enterocolita grow with and without colicin Fy in liquid cultures and compared the result. Fig. 2 shows these two cultures plated. On the left plate, the Y. enterocolitica had grown with the colicin Fy added, while the right plate is the negative control, where Y. enterocolitica had grown without any colicin Fy. If we compare the two plates, we can see that there is a clear difference in the amount of Y. enterocolitica colonies.


Read more about the Killing System

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