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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.

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.

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.

@@ Line 47: / Line 47: @@
 <h2>Bactissiles: The future of microbial combat</h2>
-<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><br>
+<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>
 <div id="p_containerM">
 <table Style="margin:15px;">
 <tr><td></td><td><h2>What we have created</h2></td></tr>
-<tr><td><img class="schedule" src="https://static.igem.org/mediawiki/2014/b/bb/Sum_up_Uppsala2014.png"</img></td>
+<tr><td><img class="schedule" src="https://static.igem.org/mediawiki/2014/e/ea/Sum_up_Uppsala14.png"</img></td>
-<td><p>Our creation, the Bactissile, is a missile bacteria, containing a two-mode system. Initially, the Bactissile will be in the <b>Target mode</b>, in which it will be producing alot of the mobility boosting gene, 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>Attacking mode</b>. In this state, the Bactissile will stop the producion of cheZ and the sRNA, due to the loss of induction and instead intiate the production of its weapon, colicin Fy. The Bactissile will then tumble around close to the target, emitting its weapon, colicin Fy. When the concentration of colicin Fy reaches a certain threshold value, the dosage will be lethal for <i>Y. enterocolitica</i>. The bacteriocin colicin Fy is only harmful for <i>Y. enterocolitica</i>, which entails that the benefitial gut flora in the Bactissiles surroundings will remain unharmed.</p></td></tr></table>
+<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>
 </div>
 <br>
@@ Line 69: / Line 69: @@
 <td>
 <p><br><i>Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:<br>
-. pSB3C17-B0032-yenbox_WT-GFP<br>
+. pSB3C17-yenbox_WT-B0032-GFP<br>
-. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR<br>
+. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23101-B0034-YenR<br>
-. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR<br>
+. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23110-B0034-YenR<br>
-. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR</i></p>
+. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23102-B0034-YenR</i></p>
 </td>
 <td>
@@ Line 80: / Line 80: @@
 </table>
-<p>By constructing the measurement 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.
+<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.
 </p>
 <br>
@@ Line 102: / Line 102: @@
 </td>
 <td>
-<p style="margin-left: 20px;"><br><i>Figure 1. A,B and C shows triplicates of swarmplate assays incubated at 2 days. 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>
+<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>
 </td>
 </tr>
 </table>
-<p>We managed to restore chemotaxis in a non-motilite mutant strain, by reintroducing the cheZ gene on plasmids into cheZ knock-out <i>E. coli</i>. The motility assay used to measure the chemotaxis was also improved by optimizing incubation conditions for cells inoculated in swarm plates. Three promoters of different strengths were tested in combination with our construct and were shown to induce different levels of motility, and hence different levels of cheZ. </p>
+<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>
 <br>
 <a href="https://2014.igem.org/Team:Uppsala/Project_Targeting">Read more about the Targeting System</a>
@@ Line 125: / Line 125: @@
 <tr>
 <td>
-<p><br><i>Figure 2. The picture show the result from an experiment to test the killing efficiency of our bacteriocin colicin Fy. On the left Y. enterocolitica had been grown in liquid culture together with colicin Fy. The right plate is a negative control, where Y. enterocolitica had grown in the same conditions in liquid culture without any colicin Fy added.</p>
+<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>
 </td>
 <td>
@@ Line 133: / Line 133: @@
 </table>
-<p>We manage to produce the bacteriocin colicin Fy and by fusing it with a His-tag we could peform a SDS-page 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 negativ 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 growing <i>Y. enterocolitica</i> colonies.
+<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.
 </p>
 <br>