Team:Uppsala/Project Killing

From 2014.igem.org

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<a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2>
<a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2>
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<p class="box_text">We wanted to use bacteriocin to design a new form of toxic that unlike general antibiotics, only kills specific pathogenes. Thereby leaving our gut flora intact. The goal was to express the bacteriocin colicin Fy in <i>E.coli</i> that is specific against <i>Y.enterocolitica</i> a pathogene found in the gut. We also wanted to include an on and off system with silencing RNA together with the sensing system. </p>
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<p class="box_text">We wanted to use a bacteriocin to design a new form of toxic that, unlike general antibiotics, only kills specific pathogenes, thereby leaving our gut flora intact. The goal was to express the bacteriocin colicin Fy in <i>E. coli</i>. Colicin Fy is specific against <i>Yersinia enterocolitica</i>, a gut pathogen. We also wanted to include an on and off system with a silencing sRNA together with the Sensing system. </p>
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<h2 class="overview">System design</h2>
<h2 class="overview">System design</h2>
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<p class="box_text">Two different constructs was needed to get the killing system to work. First the excretion tag USP45 with the bacteriocin, CFy coupled to it. Second we need a sRNA system for inhibition of the toxin when not in proximity to <i>yersinias</i>.</p>
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<p class="box_text">Two different constructs were needed to get the Killing system working. First the secretion tag, USP45, with the bacteriocin, colicin Fy, coupled to it. Secondly, we need a silencing sRNA system for inhibition of colicin Fy production when not in proximity to <i>Y. enterocolitica</i>.</p>
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<h2 class="overview">Result</h2>
<h2 class="overview">Result</h2>
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<p class="box_text">We managed to prove the existance of the bacteriocine, colicin Fy on a SDS-page. By adding lysate that contains the colicin Fy to living yersinia cultures we could also show that it works for killing the actual pathogen as well.
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<p class="box_text">We managed to prove the existance of the bacteriocin, colicin Fy on an SDS-page. By adding lysate that contains the colicin Fy to living <i>Y. enterocolitica</i> cultures we could show that it works for killing the actual pathogen as well.
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<tr><td><p>The assembly plan of the Targeting system.</p></td><td><img class= "scheduleSmall" src="https://static.igem.org/mediawiki/2014/4/4f/KillingSystem_Uppsala2014.png"></img></td></tr>
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<tr><td><p>The assembly plan of the Killing system.</p></td><td><img class= "scheduleSmall" src="https://static.igem.org/mediawiki/2014/4/4f/KillingSystem_Uppsala2014.png"></img></td></tr>
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<a id="ref_point"></a><h2>Background</h2>
<a id="ref_point"></a><h2>Background</h2>
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<h3>Bacteriocin</h3><p>With the growing problem of antibiotic resistance spreading, we decided to use bacteriocins as our antimicrobial agent. A bacteriocin is a peptide that is produced naturally by certain bacteria and targets its close relatives. Unlike general antibiotics,the target of bacteriocins are very specific. The bacteriocin, colicin Fy is produced by Y. frederiksenii and it should mostly target other yersinia species.  Bacteriocins will either attack the cell membrane or a mechanism inside  the cell such as gene expression or protein production.<sup><a href="#reference1">[1]</a></sup> One of colicins Fy main target is Y. enterocolitica. It kills Y. enterocolitica by creating pores in its cell membrane. Y. enterocolitica is also one among the common pathogens that infects the gut and cause some serious symptoms<sup><a href="#reference2">[2]</a></sup>. These were some of the reasons to choose colicin Fy as the bacteriocin we want to express.
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<h3>Bacteriocin</h3><p>With the growing problem of antibiotic resistance, we decided to use bacteriocins as our antimicrobial agent. A bacteriocin is a peptide that is produced naturally by certain bacteria and targets its close relatives. Unlike general antibiotics, the targets of bacteriocins are very specific. The bacteriocin, colicin Fy is produced by <i>Y. frederiksenii</i> and it should mostly target other yersinia species.  Bacteriocins will either attack the cell membrane or a mechanism inside  the cell, such as gene expression or protein production.<sup><a href="#reference1">[1]</a></sup> One of colicins Fy main targets is <i>Y. enterocolitica</i>. It kills <i>Y. enterocolitica</i> by creating pores in its cell membrane. <i>Y. enterocolitica</i> is also one among the common pathogens that infects the gut and causes some serious symptoms<sup><a href="#reference2">[2]</a></sup>. These were some of the reasons to choose colicin Fy as the bacteriocin to express.
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<h3>Spot42 RNA</h3><p>Our goal was to design a seek and kill system. This implies that we only want to express the colicin Fy when our bacteria is close to the target. We choose this strategy because the cell would stress out to express colicin Fy all the time, even when not in close proximity to Yersinia. In order to make our bacteria only express the colicin Fy when we want, we have designed an sRNA system. We have based our design on the system that Team Uppsala iGEM 2012 implemented.
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<h3>Spot42 sRNA</h3><p>Our goal was to design a seek and kill system. This implies that we only want to express the colicin Fy when our bacteria is close to the target. We chose this strategy because the cell would stress out to express colicin Fy all the time, even when not in close proximity to Yersinia. In order to make our bacteria express the colicin Fy only when we want it to, we have designed an sRNA system. We have based our design on the system that Team Uppsala iGEM 2012 implemented.
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. <br>The sRNA or spot42 consists of an antisense region that recognizes a specific RNA sequence to interact with and another region that recruits the protein Hfq. The Hfq protein blocks and prevents mRNA from binding to the ribosome. We have redesigned the antisense region so it will specifically recognize our USP45. This will make it bind to our USP45 and recruit the Hfq protein to stick on that region.<sup><a href="#reference3">[3]</a></sup> USP45 is the secretion tag that we have coupled to the colicin Fy. The sRNA will stick to Hfq and bind to the mRNA of USP45-Colicin Fy gene and that will block the translation.
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. <br>The sRNA, spot42, consists of an antisense region that recognizes a specific RNA sequence to interact with as well as another region that recruits the protein Hfq. The Hfq protein blocks and prevents mRNA from binding to the ribosome. We have redesigned the antisense region so it will specifically recognize our USP45. This will make it bind to our USP45 and recruit the Hfq protein to stick to that region.<sup><a href="#reference3">[3]</a></sup> USP45 is the secretion tag that we have coupled to the colicin Fy. The sRNA will stick to Hfq and bind to the mRNA of USP45-Colicin Fy gene and that will block the translation of colicin Fy.
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When our bacteria is in proximity of Y.entercolitica we want it to express the colicin Fy. Therefore, the sensing group have been working on a Yen system which will inactivate the promoter that regulates the spot42 when it is close to Y.enterocolitica.
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When our bacteria is in proximity of <i>Y. entercolitica</i> we want it to express the colicin Fy. Therefore, the Sensing group have been working on a Yen system which will inactivate the promoter that regulates the spot42 when it is close to <i>Y. enterocolitica</i>.
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<p><img class="figure2" src="https://static.igem.org/mediawiki/2014/4/41/Uppsala-igem2014Spot42_system.png"><p><i>Figure 2. The spot42 system</i></p>
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<p><img class="figure2" src="https://static.igem.org/mediawiki/2014/4/41/Uppsala-igem2014Spot42_system.png"><p><i>Figure 1. The spot42 system</i></p>
<a id="ref_point3"></a><h2>System design</h2>
<a id="ref_point3"></a><h2>System design</h2>
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<p>The aim for the killing group was to construct two different constructs- one with an export tag and the bacteriocin, CFy, and the other with a sRNA system for inhibition of the toxin when not in proximity to yersinias. However, at first we wanted to insert our final construct into lactobacillus via a shuttle vector. Because lactobacillus is a probiotic we could use our system in a real scenario if you get an Yersinia infection in the gut. The final construct would consist of J23106-B0034-USP45-CFY and a construct for the sRNA spot42. The spot42 already includes an promtor.<br>  
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<p>The aim for the killing group was to design two different constructs - one with a secretion tag and the bacteriocin, colicin Fy, and the other with an sRNA system for inhibition of the toxin when not in proximity of <i>Y. enterocolitica</i>. However, at first we wanted to insert our final construct into <i>Lactobacillus</i> via a shuttle vector. Since <i>Lactobacillus</i> is a probiotic we could use our system in a real scenario if one gets a <i>Y. enterocolitica</i> infection in the gut. The final construct would consist of J23106-B0034-USP45-CFY and a construct for the sRNA spot42. The sRNA spot42 already includes a promoter.<br>  
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Two different export tags were used. PelB for E.coli and USP45 for both Lactobacillus and E.coli. Two Anderson promoters were chosen, J23106 (strong) and J23116 (weak) for the colicin Fy because we wanted to find a balance, where E.coli would not die from stress (due to too high expression) or its own-produced toxin. To characterize our colicin Fy we wanted to purify our sample so we could see the protein on SDS-Page. In order to do this a His-Tag was required for an IMAC column purification. Therefore we also built the constructs with a 6X His-Tag.
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Two different secretion tags were used. PelB for <i>E. coli</i> and USP45 for both <i>Lactobacillus</i> and <i>E. coli</i>. Two Anderson promoters were chosen, J23106 (strong) and J23116 (weak) for the colicin Fy because we wanted to find a balance, where <i>E. coli</i> would not die from stress (due to too high expression) or its self-produced toxin. In order to characterize our colicin Fy we wanted to purify our sample so we could see the protein on an SDS-page. To do this, a His-Tag was required for an IMAC column purification. Therefore we also built the constructs with a 6x His-Tag.
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We designed the colicin Fy according to the Freiburg assembly standard, biobrick 25. This would allow us to fuse our protein with the export tag without having to consider the stop codon from the scar of SpeI and XbaI from the biobrick 10 standard.
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We designed the colicin Fy according to the Freiburg assembly standard, biobrick 25. This would allow us to fuse our protein with the secretion tag without having to consider the stop codon from the scar of SpeI and XbaI from the biobrick 10 standard.
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<h3>Silencing RNA system design</h3>
<h3>Silencing RNA system design</h3>
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<p>The silencing RNA system or sRNA system was also one of the primary aims for the killing group. We designed and modified the sRNA system from Team Uppsala iGEM 2012 called spot42. The spot42 was redesigned to sense our USP45 sequence and bind to it. We call our modified part, Spot42_USP45. The Spot42_USP45 system consist of an antisense region that will recognice our USP45 and it also has the part that recruits the Hfq protien that will block and inhibit the mRNA translation. The Hfq protein will stick to the antisense region, this will block the RBS from binding to the mRNA therefore inhibits the translation. We have designed three different antisense regions for our Spot42_USP45. They are all based on USP45 but with different lengths, 10, 15 and 20bp. This is to ensure that it is specific to only USP45. Otherwise it could bind to another region and inhibit a process that might be vital for the bacteria. We choose to have USP45 as our primary export tag since our initial goal was to build our system in lactobacillus. To help us with that we used knowledge from the Uppsala iGEM 2013 who worked with lactobacillus and USP45 among other things. 
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<p>The silencing RNA system or sRNA system was also one of the primary aims for the killing group. We designed and modified the sRNA system from Team Uppsala iGEM 2012, called spot42. The spot42 was redesigned to sense our USP45 sequence and bind to it. We call our modified part, Spot42_USP45. The Spot42_USP45 system consists of an antisense region that will recognize our USP45 and it also has the part that recruits the Hfq protien that will block and inhibit the mRNA translation. The Hfq protein will stick to the antisense region; this will block the RBS from binding to the mRNA and therefore inhibits the translation. We have designed three different antisense regions for our Spot42_USP45. They are all based on USP45 but with different lengths, 10, 15 and 20bp. This is to ensure that it is specific to only USP45. Otherwise it might bind to another region and inhibit a process that could be vital for the bacteria. We chose to have USP45 as our primary secretion tag since our initial goal was to build our system in <i>Lactobacillus</i>. To help us with that, we used knowledge from the Uppsala iGEM 2013 team, who worked with <i>Lactobacillus</i> and USP45 among other things.  
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<h3>The whole system design</h3>
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<p>Our two constructs the spot42_USP45 and J23106-B0034-USP45-CFY was to be coupled with the parts from the other project groups. When our bactosile is close to Y.enterocolitica the YenR system will sense the OHHL molecule.This will inhibit the yenbox system and thereby inhibit the CheZ and Spot42_USP45. This will make the bacteria stop and since the Spot42_USP45 is inhibited, the colicin Fy will be produced and it will be in attack mode. And when the bactisile is not in proximity of the OHHL the yenbox system will be active and it will produce CheZ and Spot42_USP45 and make the bactisile motile and not producing any colicin Fy making it be in tracking mode. See figure 3.
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<h3>Characterization</h3>
<h3>Characterization</h3>
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<p>Our initial plan was to have an secretion-tag together with our colicin Fy allowing it to secret from the living cells. Unfortunately we did not manage to get the assembly of any of the constructs to fully work. Our enzymes for the Freiburg standard did not seem to work so we had to use SpeI and XbaI causing a stop-codon that we did not managed to mutagenise away.  
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<p>Our initial plan was to have an secretion tag together with our colicin Fy allowing it to secret from the living cells. Unfortunately we did not manage to get the assembly of any of the constructs to fully work. Our enzymes for the Freiburg standard did not seem to work so we had to use SpeI and XbaI causing a stop codon that we did not managed to mutagenise away.  
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We had however succeeded in creating construct without secretion-tag. The expression of colicin Fy with x6 HIS-tag was characterized via an purification of the protein with IMAC followed by an SDS page to confirm lenght, <a href=”https://2014.igem.org/Team:Uppsala/Project_Notebook”>[Link to protocol]</a>. 11 tubes of eluent from the IMAC was saved. The protein concentration in each tube was analyzed with a nanodrop. Tube 4 and 6 had higher concentration than the other tubes and was run on SDS page.
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We had however succeeded in creating construct without secretion tag. The expression of colicin Fy with x6 His-tag was characterized via an purification of the protein with IMAC followed by an SDS-page to confirm lenght, <a href="https://2014.igem.org/Team:Uppsala/Project_Notebook">[Link to protocol]</a>. 11 tubes of eluent from the IMAC was saved. The protein concentration in each tube was analyzed with a nanodrop. Tube 4 and 6 had higher concentrations than the other tubes and were run on SDS-page.
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The results from the characterization experiments can be seen in figure 4 and 5. According to calculations based on the nucleotide sequence, the mass of colicin Fy is about 49,6 kDa. The protein ladder we used is Thermo Scientifics PageRuler Unstained Protein Ladder #26614 (figure 3). The thickest band is 50 kDa so our bands should be about at the same height.
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The results from the characterization experiments can be seen in fig. 2 and 3. According to calculations based on the nucleotide sequence, the mass of colicin Fy is about 49,6 kDa. The protein ladder we used was Thermo Scientifics PageRuler Unstained Protein Ladder #26614 (fig. 2). The thickest band is 50 kDa so our bands should be at about the same height.
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<td><img style="height:300px" src="https://static.igem.org/mediawiki/2014/0/02/Uppsala-igem2014-SDS_ladder.png"></td>
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<td><img style="height:300px; display:block; margin-right: auto; margin-left: auto;" src="https://static.igem.org/mediawiki/2014/0/02/Uppsala-igem2014-SDS_ladder.png"></td>
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<td></i>Figure 3: Thermo Scientifics PageRuler Unstained Protein Ladder #26614</i>
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<td></i>Figure 2: Thermo Scientifics PageRuler Unstained Protein Ladder #26614</i>
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<td><img src="https://static.igem.org/mediawiki/2014/1/1f/Uppsala-igem2014-SDS-page.png"></td>
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<td><i>Figure 4: The gel contains the following from the left to the right
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<td><i style="margin-left: 20px;">Figure 3: The gel contains the following from the left to the right
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<ul style="margin-left: 20px;">
<li>1. Protein ladder</li>
<li>1. Protein ladder</li>
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<li>2. Eluent 1 for lysate produced by bacteriocin producing bacteria</li>
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<li>2. Eluent 1 for lysate produced by colicin Fy producing bacteria</li>
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<li>3. Eluent 3 for lysate produced by bacteriocin producing bacteria</li>
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<li>3. Eluent 3 for lysate produced by colicin Fy producing bacteria</li>
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<li>4. Eluent 2 for lysate produced by bacteriocin producing bacteria</li>
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<li>4. Eluent 2 for lysate produced by colicin Fy producing bacteria</li>
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<li>5. Final eluent test-tube 4 for lysate produced by bacteriocin producing bacteria</li>
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<li>5. Final eluent test tube 4 for lysate produced by colicin Fy producing bacteria</li>
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<li>6. Final eluent test-tube 6 for lysate produced by bacteriocin producing bacteria</li>
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<li>6. Final eluent test tube 6 for lysate produced by colicin Fy producing bacteria</li>
<li>7. Eluent 2 for lysate produced by the negative control</li>
<li>7. Eluent 2 for lysate produced by the negative control</li>
<li>8. Eluent 3 for lysate produced by the negative control</li>
<li>8. Eluent 3 for lysate produced by the negative control</li>
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<li>9. Final eluent test-tube 4 for lysate produced by the negative control</li>
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<li>9. Final eluent test tube 4 for lysate produced by the negative control</li>
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<li>10. Final eluent test-tube 6 for lysate produced by the negative control</li>
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<li>10. Final eluent test tube 6 for lysate produced by the negative control</li>
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<p>Several IMAC and SDS-pages were run, see (Read more). In all cases eluent 1 was found to be empty. This is not surprising because the eluent volume is so small that the proteins will not come out until eluent 2. Because of this we decided to skip eluent 1 for the negative control in the presented SDS in fig. 3. Eluent 2 consists of several proteins; every protein that does not bind to the IMAC should come out in this step. Eluent 3 seems to be where our colicin Fy is eluted, since it shows a band at the correct length, 50kDa. The negative control did not show any band in 50kDa and expression could therefore be confirmed. Since the colicin was eluted at elution 3 no band can be seen at elution 4.
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  <h2>Read more/Hide</h2>
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<p>Appendix 1 displays an earlier SDS-page that was run with more constructs. Since there were more constructs we could not try all elutes, since they did not fit on one gel. The existence of colicin Fy in the construct with J23106 could be seen. Due to this we continued to try further characterization with this construct (SDS-page gel before).
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<td><img style="padding-right:20px;" src="https://static.igem.org/mediawiki/2014/d/da/Uppsala-igem2014-SDS-page-appendix.png"></td>
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<img style="height: 336px;" src="https://static.igem.org/mediawiki/2014/6/64/Uppsala-igem2014-Imac1.png">
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<td><i>Appendix 1: The gel contains the following from the right to the left:
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<li>1. Protein ladder</li>
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<img src="https://static.igem.org/mediawiki/2014/a/aa/Uppsala-igem2014-Imac2.png" style="margin-left: 170px;">
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<li>2. Final eluent 1 with CP 11 promoter</li>
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<li>3. Final eluent 2 with CP 11 promoter</li>
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<li>4. Final eluent 1 with J23106 promoter</li>
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<li>5. Final eluent 2 with J23106 promoter</li>
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<li>6. Final eluent 1 with J23113 promoter</li>
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<li>7. Final eluent 2 with J23113 promoter</li>
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<li>8. Final eluent 1 with DH5-alpha as negative control</li>
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<li>9. Final eluent 2 with DH5-alpha as negative control</li>
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<li>10. Stage 3 eluent with promoter J23106</li>
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<td><i>Picture 1: End product from the IMAC runs, each row contains eluents from the same run distributed between 10-11 ependorph tubes.</i>
 
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<td><i style="margin-left: 170px;">Picture 2. The IMAC and how it was assembled
 
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<h3>SDS-page</h3>
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<p>A in the appendix 1 is pointing towards the 50 kDa band on the ladder. B is pointing towards the band that can be seen and that likely is the colicin Fy while C is another eluent with the same promoter also showing the colicin Fy existence. This shows that the promotor J23106 can be used to produce the colicin, while CP 11 and J23113 can not.<br><br>
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We did manage to get some indication that the CP11 promoter might have worked but the band was not strong enough to guarantee that it was actually there. There can be quite a few reasons to why the band is not stronger than it is. There is a possibility that the protein was eluted out of the system too early which also seems to be the case to some extent. Appendix 1 lane 3 shows a band in the right position. This was not observed for the other tests but since that eluent is in a falcon tube containing about 40 ml of liquid it was probably very diluted and weak. It could also be some leakage from the well next to it.</p>
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The IMAC and SDS-page managed to provide a strong indication that we did produce the colicin Fy. There existed bands at the right places and the negative control did not have any bands at all. All experiments were conducted in the same manner and if it was another protein that by chance bonds to the IMAC gel it should have been in the negative control as well.
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Most bacteriocins are not fully studied so there might always be unknown problems with them. One possibility is that they somehow hurt the bacteria in which they are produced. Since the colicin described here is supposed to only react to a specific receptor that some <i>Yersinia</i> species have on the cell surface the probability for the colicin to hurt the producing bacteria is very low. A bigger problem could be that many bacteriocins are short lived in certain conditions and might denature or break before they are characterized.</p>
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<img src="https://static.igem.org/mediawiki/2014/a/a0/Uppsala-igem2014-SDS.png">
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<h3>Inhibition test</h3>
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<p><i>Figure 3. SDS-page during the run</i></p>
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<p>To be able to prove that our system is working we needed to test it on the actual <i>Y. enterocolitica</i>. Since <i>Y. enterocolitica</i> is a class two bacteria we had to do it in another lab. We managed to get in contact with Livsmedelsverket (the Swedish authority of food safety) and they allowed us to test our colicin Fy in one of their labs on one of their pathogenic strains of <i>Y. enterocolitica</i>.
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To examine the inhibition of colicin Fy an overnight culture of bacteriocin producing bacteria in 200ml LB with no antibiotic was prepared. As negative control, DH5-alpha was grown under the same conditions. No antibiotic was added to ensure that the antibiotics would not affect the results. The cells were then lysed and the supernatant was collected according to <a href="https://2014.igem.org/Team:Uppsala/Project_Notebook">protocol</a>. The supernatant was added to a final concentration of 1%(v/v) in LB with 10^4 CFU/ml <i>Y. enterocolitica</i> added. The culture was put to grow in 37d C with 100rpm shaking. OD measurements were taken every hour,  but due to <i>Y. enterocolitica</i> ability to aggregate the results were inconclusive. Instead 100microL was plated on BHI agar plates after 0h, 2h, 4h and 24h. Immediately growth difference could be spotted and after 4 hours the results were very clear, fig. 4. More figures of the plating can be found in the appendix.</p>
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<p>After having purified the protein with the help of IMAC the next step is to show that it is actually there. SDS-page in theory works like a normal gel electrophoresis but it is for proteins instead of nucleic acid. Proteins get a similar charge and structure due to the substances SDS, DTT as well as heating it to 95 degrees, centigrade for 5 minutes and thereby it is possible to distinguish them by size alone. By having a protein ladder next to it, it is possible to see what size the other proteins in the gel are.</p>
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<p>The results from the characterization experiments can be seen in figurepicture 5 and 6. According to calculations based on the nucleotide sequence the mass of the colicin Fybacteriocin is about 49,6 KDa. the protein ladder we used is thermo scientifics PageRuler Unstained Protein Ladder #26614 (figurepicture 4). As seen in the picture the biggest band is 50 kDa so our bands should be at the same height.
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<td><img style="width: 600px; padding-right: 20px" src="https://static.igem.org/mediawiki/2014/5/55/Uppsala-igem2014-4h_10.jpg"</td>
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<img src="https://static.igem.org/mediawiki/2014/1/1f/Uppsala-igem2014-SDS-page.png">
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<td><i>Figure 4: Tests to compare colicin Fy lysate on the right plate compared to the N/C on the left. Both plates are diluted to the same concentration and grown for the same amount of time. For more figures, see "Read more"</i></td>
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<img src="https://static.igem.org/mediawiki/2014/0/02/Uppsala-igem2014-SDS_ladder.png">
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-
</td>
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-
</tr>
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-
<tr>
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-
<td>Picture 4: The gel contains the following from the left to the right<br>
+
-
Protein ladder<br>
+
-
eluent 1 for lysate produced by bacteriocin producing bacteria<br>
+
-
eluent 3 for lysate produced by bacteriocin producing bacteria<br>
+
-
eluent 2 for lysate produced by bacteriocin producing bacteria<br>
+
-
Final eluent testtube 4 for lysate produced by bacteriocin producing bacteria<br>
+
-
Final eluent testtube 6 for lysate produced by bacteriocin producing bacteria<br>
+
-
eluent 2 for lysate produced by the negative control<br>
+
-
eluent 3 for lysate produced by the negative control<br>
+
-
final eluent testtube 4 for lysate produced by the negative control<br>
+
-
Final eluent testube 6 for lysate produced by the negative control<br>
+
-
</td>
+
</tr>
</tr>
</table>
</table>
-
 
-
</div>
 
-
 
-
<p>Eluent 1 is empty, this might at first be surprising but the volume added is so smal that the free proteins not sticking will not come out until eluent 2. because of this we decided to skip eluent 1 for the negative controll. Eluent 2 is comprised of lots of different proteins, everything not sticking to the gel should come out in this step. there was an error in the loading of the samples so eluent 2 and 3 have changed place compred to what is locigal. Eluent 3 seems to be when our colicin Fybacteriocin was eluated seeing it showed a band at the correct length. The negative controll did not show any band in this spot. Seeing the protein was eluated here there wasn´t anything left to get out on the higest concentraion.
 
-
</p>
 
 +
<br><br>
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   <h2>Read more/Hide</h2>
    
    
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<p>On all of the following nine pictures the plate to the right is the negative control while the bacterias with colicin Fy added is to the left. The first three pictures shows 3 different dilutions, all done at time zero. They are in the order, not diluted, diluted 10 times and diluted 100 times. The three pictures after that are diluted in the order of 10, 100 and lastly 1000 times , they are all plated after 2 hours. The last 3 plates are also diluted, 10, 100 and lastly 1000 times and are </p>
-
<p>A picture of an earlier SDS-page that was run with more constructs, due to this we didn´t have that many controls to prove that this was the case and we couldn´t try all eluates. The existance of colicin Fybacteriocin in the construct with j23106 could be seen though, due to this we continued to try further charectarization with this construct (SDS-page gel before).
+
<img style="width: 1024px;" src="https://static.igem.org/mediawiki/2014/4/49/Uppsala-igem2014-livs.jpg"><i>Right: Negative control, Left: Colicin Fy, time 0 hour, not diluted</i>  
-
</p>
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-
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-
<img src="https://static.igem.org/mediawiki/2014/d/da/Uppsala-igem2014-SDS-page-appendix.png">
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-
<i>Picture 5: The gel contains the following from the right to the left:<br>
+
-
Protein ladder<br>
+
-
Final eluent 1 with CP 11 promoter<br>
+
-
Final eluent 2 with CP 11 promoter<br>
+
-
Final eluent 1 with J23106 promoter<br>
+
-
Final eluent 2 with J23106 promoter<br>
+
-
Final eluent 1 with j23113 promoter<br>
+
-
Final eluent 2 with J23113 promoter<br>
+
-
Final eluent 1 with DH5-alpha as negative control<br>
+
-
Final eluent 2 with DH5-alpha as negative control<br>
+
-
Stage 3 eluent with promoter j23106<br>
+
-
</i>
+
-
<p>A in the picture is pointing towards the 50kDa band on the ladder. B is pointing towards the band that can be seen and that likely is the colicin Fybacteriocin while C is another eluent with the same promoter also showing the existance
+
<br><br>
-
</p>
+
<img src="https://static.igem.org/mediawiki/2014/0/0b/Uppsala-igem2014-livs2.jpg"><i>Right: Negative control, Left: Colicin Fy, time 0 hour, diluted 1:10</i>
 +
 
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/1/17/Uppsala-igem2014-livs3.jpg"> <i>Right: Negative control, Left: Colicin Fy, time 0 hour, diluted 1:100</i>
 +
 
 +
<br><br>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/b/bd/Uppsala-igem2014-livs4.jpg"><i>Right: Negative control, Left: Colicin Fy, time 2 hour, diluted 1:10</i>
 +
 
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/e/ef/Uppsala-igem2014-livs5.jpg"> <i>Right: Negative control, Left: Colicin Fy, time 2 hour, diluted 1:100</i>
 +
 
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d1/Uppsala-igem2014-livs6.jpg"> <i>Right: Negative control, Left: Colicin Fy, time 2 hour, diluted 1:1000</i>
 +
 
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/e/e6/Uppsala-igem2014-livs7.jpg"> <i>Right: Negative control, Left: Colicin Fy, time 4 hour, diluted 1:10</i>
 +
 
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/c/cf/Uppsala-igem2014-livs8.jpg"> <i>Right: Negative control, Left: Colicin Fy, time 4 hour, diluted 1:100</i>
 +
<br><br>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/5/53/Uppsala-igem2014-livs9.jpg"><br> <i>Right: Negative control, Left: Colicin Fy, time 4 hour, diluted 1:1000</i>
 +
<br><p>As seen in all the pictures the bacterias with colicin Fy are less or about the same amount of bacteria in all the pictures.</p><br><br><p>The following 4 pictures are all Y.enterocoliticas plated on Yersinia specific plates to make sure that it isn´t a contamination that we see. All the plates in the first three pictures have all been dilluted 10-1while the plates on the last picture was diluted 10-4. </p>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/c/c3/Uppsala-igem2014-livs10.jpg"><br>
 +
<i>Plates at time zero. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control.</i>
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/b/b4/Uppsala-igem2014-livs11.jpg"><br>
 +
<i>Plates after two hours. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control</i>
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/9/9f/Uppsala-igem2014-livs12.jpg"><br>
 +
<i>Plates after 4 hours. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control</i>
 +
<br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d4/Uppsala-igem2014-livs13.jpg">
 +
<i>Plates after 26 hour. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control</i><br><br>
 +
<p>In conclusion colicin Fy inhibits the growth of yersinias very fast and the effects lasts for a long while acording to the results, the negative control always grows much more.</p>
  
  
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<p>The IMAC and SDS-page managed to provide a strong indication that we did produce the colicin Fybacteriocin. There existed bands at the right places and the negative control did not have any bands at all. All the experiments were conducted in the same manor and if it was another protein that by chance stuck to the IMAC gel it should have been in the negative control as well. Regarding the different promoters we used J23106 ,is a well-studiedwell studied promoter and it is the one that is the clearest that it is actully there.
 
<br><br>
<br><br>
-
We did managed to get some indication that also the CP11 promoter might also have worked but the band wasn´t strong enough to be sure. There can be quite a few reasons to why the band isn´t stronger than it is. Apart from the histidine tag posibly having an inhibiting efect Tthere is also a possibility that the protein was eluted out of the system too early which also seems to be the case to some extent. FigurePicture 5 lane 3 shows a band in the right position, this was not observed for the other tests but since that eluent is in a falcon tube containing about 40 ml of liquid it was probably very diluted and weak. It coulds also only be some leakage from the well next to it.
+
 
-
<br><br>
+
 
-
Most Bacteriocins aren´t fully studied so there might always be unknown troubles with them, one possibility is that they somehow hurt the bacteria in which they areit is produced, in. Bbut since the colicin described here is supposed to only react to a specific receptor that some Yersinia. E haves on the cell surface the chance for that is probably very low. A bigger problem could be that many Bacteriocins are short lived in certain conditions and might denature or break before they are characterized. Another factor that might interfere with the expression of the protein is that according to some articles a Histidine tag at the C-terminal can inhibit the expression as much as to only produce a tenth of the original production level.
+
 
-
</p>
+
 
 +
 
 +
 
<h3>Colicin Fy specificity test</h3>
<h3>Colicin Fy specificity test</h3>
-
<p>To se that Colicin Fy realy is as specific as we though we had to try it on other bacterias. We  Apart from testing them on Y. enterocoliticas we also managed to get ahold of clas 1 versions of pseudumonas, enterobacter, klebsiella as well as lactobacilus plantarum and the standard E. coli DH5-alpha. We did multiple test with varying results. During the first experiment apart from plating the cultures we also measured the absorption of liquid cultures at OD value 600. The results can be seen in the table below, it indicates that the colicin Fy is probably specifik but something seems to have gone wrong with the plating and the result is inconclusive.
+
<p>To see if the Colicin Fy really is as specific as we thought, we had to try it on other bacteria. Apart from testing them on <i>Y. enterocolitica</i> we also managed to get a hold of risk class one versions of <i>Pseudomonas</i>, <i>Enterobacter</i>, <i>Klebsiella</i> as well as <i>Lactobacillus plantarum</i> and the standard <i>E. coli</i> DH5-alpha. We did multiple tests with varying results. The tests were performed in the same way as the inhibition test above. However OD600 measurements were made here since no aggregations could be seen in most of our cultures, see table A. The table indicates that the colicin Fy is probably specific, but something seems to have gone wrong with the plating, see figures E, and that result was inconclusive.<p>
-
</p>
+
<h3>OD value after 2H</h3>
<h3>OD value after 2H</h3>
Line 240: Line 249:
<th></th>
<th></th>
<th>DH5-alpha</th>
<th>DH5-alpha</th>
-
<th>pseudomonas</th>
+
<th>Pseudomonas</th>
-
<th>enterobacter</th>
+
<th>Enterobacter</th>
-
<th>klebsiella</th>
+
<th>Klebsiella</th>
-
<th>lactobacillus</th>
+
<th>Lactobacillus</th>
</tr>
</tr>
<tr>
<tr>
-
<td>Controll</td>
+
<td>Control</td>
<td>0.390</td>
<td>0.390</td>
<td>0.085</td>
<td>0.085</td>
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<th></th>
<th></th>
<th>DH5-alpha</th>
<th>DH5-alpha</th>
-
<th>pseudomonas</th>
+
<th>Pseudomonas</th>
-
<th>enterobacter</th>
+
<th>Enterobacter</th>
-
<th>klebsiella</th>
+
<th>Klebsiella</th>
-
<th>lactobacillus</th>
+
<th>Lactobacillus</th>
</tr>
</tr>
<tr>
<tr>
-
<td>Controll</td>
+
<td>Control</td>
<td>0.520</td>
<td>0.520</td>
<td>0.137</td>
<td>0.137</td>
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<th></th>
<th></th>
<th>DH5-alpha</th>
<th>DH5-alpha</th>
-
<th>pseudomonas</th>
+
<th>Pseudomonas</th>
-
<th>enterobacter</th>
+
<th>Enterobacter</th>
-
<th>klebsiella</th>
+
<th>Klebsiella</th>
-
<th>lactobacillus</th>
+
<th>Lactobacillus</th>
</tr>
</tr>
<tr>
<tr>
-
<td>Controll</td>
+
<td>Control</td>
<td>1.003**</td>
<td>1.003**</td>
<td>1.903</td>
<td>1.903</td>
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</table>
</table>
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<p><i> *The broth used to grow the lactobacillus bacteria wasn´t available to calibrate the spectrophotometer with. Therefore the values will be wrong but the comparison betwen the control and the colicin Fy should still be valid.<br>
+
<p><i> *The broth used to grow the lactobacillus bacteria was not available to calibrate the spectrophotometer. Therefore the values will be wrong but the comparison between the control and the colicin Fy should still be valid.<br>
-
**The bacteria had created precipations which makes the measurings unreliable.
+
**The bacteria had created precipations which made the measurings unreliable.
</i></p>
</i></p>
 +
<br><br>
 +
<img style="width: 100%" src="https://static.igem.org/mediawiki/2014/c/c1/Uppsala-igem2014killing5.jpg">
 +
<i>Figure 5: Specificity inhibition test. Top row is control plates, bottom row is with colicin. From left to right <i>Lactobacillus Plantarum</i>, <i>Klebsiella</i>, <i>E. coli</i>,  <i>Pseudomonas</i>, <i>Enterobacter</i>.</i>
 +
<br><br>
 +
<p>Since the experiment did not give any clear results we did the experiment again with new lysates, one with colicin Fy and one negative control. At first it seemed as if the colicin Fy killed most of them but at a closer look we saw two types of colonies on some of the plates, probably contamination. When plating out the lysates that should be sterile, some bacteria still grew, but they grew much more in the controls. If the contaminated plates are ignored the colicin Fy seems specific, but more experiments need to be done to be sure. Due to lack of time this was not possible.</p>
-
<h3>Livsmedelsverket (Food administration)</h3>
+
<img src="https://static.igem.org/mediawiki/2014/8/8e/Uppsala-igem2014killing6.jpg">
-
 
+
<i>Figure 6: The E on the plates stands for Enterobacter, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. the numbers 10, 100 and 300 stands for the amount of µl of lysate added.</i>
-
<p>To be able to  prove that our system is working we needed to test it on the actual dangerous bacteria. Since Y.entrecolitica is a class two2 bacteria we had to do it in another lab. We managed to get in contact with Livsmedelsverket (the Swedish autorithy of food safety) and they allowed us to test our colicin Fybacteriocin in one of their labs.
+
<br><br>
<br><br>
-
Our initial plan was to have an export-tag together with our colicin Fybacteriocin allowing it to come out from the living cells. Unfortunately we didn´t manage to get any of the constructs to fully work. We designed the colicin Fybacteriocin according to the Freiburg assembly standard, biobrick 25. This would allow us to fuse our protein with the export tag without having to consider the stop codon from the scar of SpeI and XbaI from the biobrick 10 standards. Oour enzymes did not seem to work however so we had to use SpeI and XbaI causing a stop-codon that we did not managed to mutagenise away.
+
<img src="https://static.igem.org/mediawiki/2014/0/09/Uppsala-igem2014killing7.jpg">
 +
<i>Figure 7: Controls of the lysate to see if they were contaminated or not, to the left is the control lysate while the bacteriocin is to the right.</i>
<br><br>
<br><br>
-
What we did manage to do though was, as seen from the SDS-page, to show that the colicin Fybacteriocin was actually expressed. To get the colicin Fybacteriocin out of the cell we lysate it by sonication [    länk till protokol!!!  ] and after that centrifugedcentrifugated it at max speed to get rid of cell organelles and other bigger things. To see that it truly is the colicin Fybacteriocin, we grew two2 cultures under the same conditions and then added the same amounts and concentrations of them to liquid cultures. After that we plated them during different times to compare the differences. All tests showed that the colicin Fybacteriocin always killed more yesrsinas than the negative controll.  
+
<p>As seen in fig. 7 the control lysate is much more contaminated than the bacteriocin, this explains why the control plates in all cases have more bacteria on the plates plated with 300 µl of lysate added compared with all the others. Different types of colonies can also be seen on most of them.  
 +
<br>
 +
In conclusion there are some indications that colicin Fy is specific but more tests need to be done.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2014/5/55/Uppsala-igem2014-4h_10.jpg">
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-
<p><i>picture 7: Tests to compare colicin Fybacteriocin lysate on the right plate compared to the N/C on the left, both plates are diluted to the same concentration as taken at the same time, for more picture check the APENDIX?
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</i></p>
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  <h2>Read more/Hide</h2>
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<img src="https://static.igem.org/mediawiki/2014/3/36/Uppsala-igem2014KillingA2.jpg">
 +
<i>Appendix 2: The K on the plates stands for klebsiella, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. the numbers 10, 100 and 300 stands for the amount of µl of lysate added.</i>
 +
<br><br>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/7/73/Uppsala-igem2014KillingA3.jpg">
 +
<i>Appendix 3: The D on the plates stands for DH5-alpha (E. coli), the C stands for control lysate added while B for bacteriocin (colicin Fy) added. The numbers 10, 100 and 300 stands for the amount of µl of lysate added.</i>
 +
<br><br>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2014/0/0b/Uppsala-igem2014KillingA4.jpg">
 +
<i>Appendix 4: The P on the plates stands for pseudomonas, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. The numbers 10, 100 and 300 stands for the amount of µl of lysate added.</i>
 +
 
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<a id="ref_point4"></a><h2>Parts</h2>
<a id="ref_point4"></a><h2>Parts</h2>
<table id=partsT style="width:100%">
<table id=partsT style="width:100%">
-
<tr><th></th><th>BioBrick code</th><th>Type</th><th>Construct</th><th>Description</th><th>Designers</th></tr>
+
<tr><th>Fav.</th><th>BioBrick code</th><th>Type</th><th>Construct</th><th>Description</th><th>Designers</th></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381014">BBa_K1381014</a></td><td>Regulatory</td><td>J23101-spot42</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381014">BBa_K1381014</a></td><td>Regulatory</td><td>J23101-spot42_USP45_10bp</td><td>Silencing sRNA with affinity to USP45</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381015">BBa_K1381015</a></td><td>Regulatory</td><td>J23101-spot42</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381015">BBa_K1381015</a></td><td>Regulatory</td><td>J23101-spot42_USP45_15bp</td><td>Silencing sRNA with affinity to USP45</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381016">BBa_K1381016</a></td><td>Regulatory</td><td>J23101-spot42</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381016">BBa_K1381016</a></td><td>Regulatory</td><td>J23101-spot42_USP45_20bp</td><td>Silencing sRNA with affinity to USP45</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381017">BBa_K1381017</a></td><td>Coding</td><td>CFY-X6 his</td><td></td><td>Killing Group</td></tr>
+
<tr><td><img src="https://static.igem.org/mediawiki/2014/e/ef/GoodGuy.png" Style="width: 20px"></img></td><td><a href="http://parts.igem.org/Part:BBa_K1381017">BBa_K1381017</a></td><td>Coding</td><td>CFY-X6 his</td><td>The gene coding for the bacteriocin colicin Fy</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381018">BBa_K1381018</a></td><td>Tag</td><td>B0034-USP45</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381018">BBa_K1381018</a></td><td>Tag</td><td>B0034-USP45</td><td>The secretion tag USP45 with the RBS B0034</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381019">BBa_K1381019</a></td><td>Device</td><td>B0034-pelB-CFY-X6 his</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381019">BBa_K1381019</a></td><td>Device</td><td>B0034-pelB-CFY-X6 his</td><td>The bacteriocin colicin Fy coupled to the the secretion tag pelB</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381020">BBa_K1381020</a></td><td>Generator</td><td>J23106-B0034-pelB-CFY-X6 his</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381020">BBa_K1381020</a></td><td>Generator</td><td>J23106-B0034-pelB-CFY-X6 his</td><td>Colicin Fy coupled to the the secretion tag pelB with the promoter J23106</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381021">BBa_K1381021</a></td><td>Generator</td><td>J23116-B0034-pelB-CFY-X6 his</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381021">BBa_K1381021</a></td><td>Generator</td><td>J23116-B0034-pelB-CFY-X6 his</td><td>Colicin Fy coupled to the the secretion tag pelB with the promoter J23116</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381022">BBa_K1381022</a></td><td>Device</td><td>B0034-USP45-CFY-X6 his</td><td></td><td>Killing Group</td></tr>
+
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381022">BBa_K1381022</a></td><td>Device</td><td>B0034-USP45-CFY-X6 his</td><td>The bacteriocin colicin Fy coupled to the the secretion tag USP45</td><td>Killing Group</td></tr>
-
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381023">BBa_K1381023</a></td><td>Generator</td><td>J23106-B0034-CFY-X6 his</td><td></td><td>Killing Group</td></tr>
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<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381023">BBa_K1381023</a></td><td>Generator</td><td>J23106-B0034-CFY-X6 his</td><td>Colicin Fy coupled to the promoter J23106</td><td>Killing Group</td></tr>
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Latest revision as of 21:40, 17 October 2014

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The assembly plan of the Killing system.

Background

Bacteriocin

With the growing problem of antibiotic resistance, we decided to use bacteriocins as our antimicrobial agent. A bacteriocin is a peptide that is produced naturally by certain bacteria and targets its close relatives. Unlike general antibiotics, the targets of bacteriocins are very specific. The bacteriocin, colicin Fy is produced by Y. frederiksenii and it should mostly target other yersinia species. Bacteriocins will either attack the cell membrane or a mechanism inside the cell, such as gene expression or protein production.[1] One of colicins Fy main targets is Y. enterocolitica. It kills Y. enterocolitica by creating pores in its cell membrane. Y. enterocolitica is also one among the common pathogens that infects the gut and causes some serious symptoms[2]. These were some of the reasons to choose colicin Fy as the bacteriocin to express.

Spot42 sRNA

Our goal was to design a seek and kill system. This implies that we only want to express the colicin Fy when our bacteria is close to the target. We chose this strategy because the cell would stress out to express colicin Fy all the time, even when not in close proximity to Yersinia. In order to make our bacteria express the colicin Fy only when we want it to, we have designed an sRNA system. We have based our design on the system that Team Uppsala iGEM 2012 implemented. .
The sRNA, spot42, consists of an antisense region that recognizes a specific RNA sequence to interact with as well as another region that recruits the protein Hfq. The Hfq protein blocks and prevents mRNA from binding to the ribosome. We have redesigned the antisense region so it will specifically recognize our USP45. This will make it bind to our USP45 and recruit the Hfq protein to stick to that region.[3] USP45 is the secretion tag that we have coupled to the colicin Fy. The sRNA will stick to Hfq and bind to the mRNA of USP45-Colicin Fy gene and that will block the translation of colicin Fy. When our bacteria is in proximity of Y. entercolitica we want it to express the colicin Fy. Therefore, the Sensing group have been working on a Yen system which will inactivate the promoter that regulates the spot42 when it is close to Y. enterocolitica.

Figure 1. The spot42 system

System design

The aim for the killing group was to design two different constructs - one with a secretion tag and the bacteriocin, colicin Fy, and the other with an sRNA system for inhibition of the toxin when not in proximity of Y. enterocolitica. However, at first we wanted to insert our final construct into Lactobacillus via a shuttle vector. Since Lactobacillus is a probiotic we could use our system in a real scenario if one gets a Y. enterocolitica infection in the gut. The final construct would consist of J23106-B0034-USP45-CFY and a construct for the sRNA spot42. The sRNA spot42 already includes a promoter.
Two different secretion tags were used. PelB for E. coli and USP45 for both Lactobacillus and E. coli. Two Anderson promoters were chosen, J23106 (strong) and J23116 (weak) for the colicin Fy because we wanted to find a balance, where E. coli would not die from stress (due to too high expression) or its self-produced toxin. In order to characterize our colicin Fy we wanted to purify our sample so we could see the protein on an SDS-page. To do this, a His-Tag was required for an IMAC column purification. Therefore we also built the constructs with a 6x His-Tag.

We designed the colicin Fy according to the Freiburg assembly standard, biobrick 25. This would allow us to fuse our protein with the secretion tag without having to consider the stop codon from the scar of SpeI and XbaI from the biobrick 10 standard.

Silencing RNA system design

The silencing RNA system or sRNA system was also one of the primary aims for the killing group. We designed and modified the sRNA system from Team Uppsala iGEM 2012, called spot42. The spot42 was redesigned to sense our USP45 sequence and bind to it. We call our modified part, Spot42_USP45. The Spot42_USP45 system consists of an antisense region that will recognize our USP45 and it also has the part that recruits the Hfq protien that will block and inhibit the mRNA translation. The Hfq protein will stick to the antisense region; this will block the RBS from binding to the mRNA and therefore inhibits the translation. We have designed three different antisense regions for our Spot42_USP45. They are all based on USP45 but with different lengths, 10, 15 and 20bp. This is to ensure that it is specific to only USP45. Otherwise it might bind to another region and inhibit a process that could be vital for the bacteria. We chose to have USP45 as our primary secretion tag since our initial goal was to build our system in Lactobacillus. To help us with that, we used knowledge from the Uppsala iGEM 2013 team, who worked with Lactobacillus and USP45 among other things.

Result

Characterization

Our initial plan was to have an secretion tag together with our colicin Fy allowing it to secret from the living cells. Unfortunately we did not manage to get the assembly of any of the constructs to fully work. Our enzymes for the Freiburg standard did not seem to work so we had to use SpeI and XbaI causing a stop codon that we did not managed to mutagenise away.

We had however succeeded in creating construct without secretion tag. The expression of colicin Fy with x6 His-tag was characterized via an purification of the protein with IMAC followed by an SDS-page to confirm lenght, [Link to protocol]. 11 tubes of eluent from the IMAC was saved. The protein concentration in each tube was analyzed with a nanodrop. Tube 4 and 6 had higher concentrations than the other tubes and were run on SDS-page.

The results from the characterization experiments can be seen in fig. 2 and 3. According to calculations based on the nucleotide sequence, the mass of colicin Fy is about 49,6 kDa. The protein ladder we used was Thermo Scientifics PageRuler Unstained Protein Ladder #26614 (fig. 2). The thickest band is 50 kDa so our bands should be at about the same height.

Figure 2: Thermo Scientifics PageRuler Unstained Protein Ladder #26614
Figure 3: The gel contains the following from the left to the right
  • 1. Protein ladder
  • 2. Eluent 1 for lysate produced by colicin Fy producing bacteria
  • 3. Eluent 3 for lysate produced by colicin Fy producing bacteria
  • 4. Eluent 2 for lysate produced by colicin Fy producing bacteria
  • 5. Final eluent test tube 4 for lysate produced by colicin Fy producing bacteria
  • 6. Final eluent test tube 6 for lysate produced by colicin Fy producing bacteria
  • 7. Eluent 2 for lysate produced by the negative control
  • 8. Eluent 3 for lysate produced by the negative control
  • 9. Final eluent test tube 4 for lysate produced by the negative control
  • 10. Final eluent test tube 6 for lysate produced by the negative control

Several IMAC and SDS-pages were run, see (Read more). In all cases eluent 1 was found to be empty. This is not surprising because the eluent volume is so small that the proteins will not come out until eluent 2. Because of this we decided to skip eluent 1 for the negative control in the presented SDS in fig. 3. Eluent 2 consists of several proteins; every protein that does not bind to the IMAC should come out in this step. Eluent 3 seems to be where our colicin Fy is eluted, since it shows a band at the correct length, 50kDa. The negative control did not show any band in 50kDa and expression could therefore be confirmed. Since the colicin was eluted at elution 3 no band can be seen at elution 4.



Read more/Hide

Appendix 1 displays an earlier SDS-page that was run with more constructs. Since there were more constructs we could not try all elutes, since they did not fit on one gel. The existence of colicin Fy in the construct with J23106 could be seen. Due to this we continued to try further characterization with this construct (SDS-page gel before).

Appendix 1: The gel contains the following from the right to the left:
  • 1. Protein ladder
  • 2. Final eluent 1 with CP 11 promoter
  • 3. Final eluent 2 with CP 11 promoter
  • 4. Final eluent 1 with J23106 promoter
  • 5. Final eluent 2 with J23106 promoter
  • 6. Final eluent 1 with J23113 promoter
  • 7. Final eluent 2 with J23113 promoter
  • 8. Final eluent 1 with DH5-alpha as negative control
  • 9. Final eluent 2 with DH5-alpha as negative control
  • 10. Stage 3 eluent with promoter J23106


A in the appendix 1 is pointing towards the 50 kDa band on the ladder. B is pointing towards the band that can be seen and that likely is the colicin Fy while C is another eluent with the same promoter also showing the colicin Fy existence. This shows that the promotor J23106 can be used to produce the colicin, while CP 11 and J23113 can not.

We did manage to get some indication that the CP11 promoter might have worked but the band was not strong enough to guarantee that it was actually there. There can be quite a few reasons to why the band is not stronger than it is. There is a possibility that the protein was eluted out of the system too early which also seems to be the case to some extent. Appendix 1 lane 3 shows a band in the right position. This was not observed for the other tests but since that eluent is in a falcon tube containing about 40 ml of liquid it was probably very diluted and weak. It could also be some leakage from the well next to it.



The IMAC and SDS-page managed to provide a strong indication that we did produce the colicin Fy. There existed bands at the right places and the negative control did not have any bands at all. All experiments were conducted in the same manner and if it was another protein that by chance bonds to the IMAC gel it should have been in the negative control as well. Most bacteriocins are not fully studied so there might always be unknown problems with them. One possibility is that they somehow hurt the bacteria in which they are produced. Since the colicin described here is supposed to only react to a specific receptor that some Yersinia species have on the cell surface the probability for the colicin to hurt the producing bacteria is very low. A bigger problem could be that many bacteriocins are short lived in certain conditions and might denature or break before they are characterized.

Inhibition test

To be able to prove that our system is working we needed to test it on the actual Y. enterocolitica. Since Y. enterocolitica is a class two bacteria we had to do it in another lab. We managed to get in contact with Livsmedelsverket (the Swedish authority of food safety) and they allowed us to test our colicin Fy in one of their labs on one of their pathogenic strains of Y. enterocolitica.

To examine the inhibition of colicin Fy an overnight culture of bacteriocin producing bacteria in 200ml LB with no antibiotic was prepared. As negative control, DH5-alpha was grown under the same conditions. No antibiotic was added to ensure that the antibiotics would not affect the results. The cells were then lysed and the supernatant was collected according to protocol. The supernatant was added to a final concentration of 1%(v/v) in LB with 10^4 CFU/ml Y. enterocolitica added. The culture was put to grow in 37d C with 100rpm shaking. OD measurements were taken every hour, but due to Y. enterocolitica ability to aggregate the results were inconclusive. Instead 100microL was plated on BHI agar plates after 0h, 2h, 4h and 24h. Immediately growth difference could be spotted and after 4 hours the results were very clear, fig. 4. More figures of the plating can be found in the appendix.

Figure 4: Tests to compare colicin Fy lysate on the right plate compared to the N/C on the left. Both plates are diluted to the same concentration and grown for the same amount of time. For more figures, see "Read more"


Read more/Hide

On all of the following nine pictures the plate to the right is the negative control while the bacterias with colicin Fy added is to the left. The first three pictures shows 3 different dilutions, all done at time zero. They are in the order, not diluted, diluted 10 times and diluted 100 times. The three pictures after that are diluted in the order of 10, 100 and lastly 1000 times , they are all plated after 2 hours. The last 3 plates are also diluted, 10, 100 and lastly 1000 times and are

Right: Negative control, Left: Colicin Fy, time 0 hour, not diluted

Right: Negative control, Left: Colicin Fy, time 0 hour, diluted 1:10

Right: Negative control, Left: Colicin Fy, time 0 hour, diluted 1:100

Right: Negative control, Left: Colicin Fy, time 2 hour, diluted 1:10

Right: Negative control, Left: Colicin Fy, time 2 hour, diluted 1:100

Right: Negative control, Left: Colicin Fy, time 2 hour, diluted 1:1000

Right: Negative control, Left: Colicin Fy, time 4 hour, diluted 1:10

Right: Negative control, Left: Colicin Fy, time 4 hour, diluted 1:100


Right: Negative control, Left: Colicin Fy, time 4 hour, diluted 1:1000

As seen in all the pictures the bacterias with colicin Fy are less or about the same amount of bacteria in all the pictures.



The following 4 pictures are all Y.enterocoliticas plated on Yersinia specific plates to make sure that it isn´t a contamination that we see. All the plates in the first three pictures have all been dilluted 10-1while the plates on the last picture was diluted 10-4.


Plates at time zero. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control.


Plates after two hours. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control


Plates after 4 hours. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control

Plates after 26 hour. The two plates to the left are J23106-B0034-CFY on different backbones and the last one to the right is a negative control

In conclusion colicin Fy inhibits the growth of yersinias very fast and the effects lasts for a long while acording to the results, the negative control always grows much more.



Colicin Fy specificity test

To see if the Colicin Fy really is as specific as we thought, we had to try it on other bacteria. Apart from testing them on Y. enterocolitica we also managed to get a hold of risk class one versions of Pseudomonas, Enterobacter, Klebsiella as well as Lactobacillus plantarum and the standard E. coli DH5-alpha. We did multiple tests with varying results. The tests were performed in the same way as the inhibition test above. However OD600 measurements were made here since no aggregations could be seen in most of our cultures, see table A. The table indicates that the colicin Fy is probably specific, but something seems to have gone wrong with the plating, see figures E, and that result was inconclusive.

OD value after 2H

DH5-alpha Pseudomonas Enterobacter Klebsiella Lactobacillus
Control 0.390 0.085 0.620 0.563 1.100*
Colicin Fy 0.378 0.098 0.618 0.578 0.109*

OD value after 4h

DH5-alpha Pseudomonas Enterobacter Klebsiella Lactobacillus
Control 0.520 0.137 0.639 0.669 1.747*
Colicin Fy 0.503 0.139 0.658 0.748 1.815*

OD valueafter 24H

DH5-alpha Pseudomonas Enterobacter Klebsiella Lactobacillus
Control 1.003** 1.903 1.151** 1.012 2.589**
Colicin Fy 1.464** 1.990 1.825** 1.035 2.540*

*The broth used to grow the lactobacillus bacteria was not available to calibrate the spectrophotometer. Therefore the values will be wrong but the comparison between the control and the colicin Fy should still be valid.
**The bacteria had created precipations which made the measurings unreliable.



Figure 5: Specificity inhibition test. Top row is control plates, bottom row is with colicin. From left to right Lactobacillus Plantarum, Klebsiella, E. coli, Pseudomonas, Enterobacter.

Since the experiment did not give any clear results we did the experiment again with new lysates, one with colicin Fy and one negative control. At first it seemed as if the colicin Fy killed most of them but at a closer look we saw two types of colonies on some of the plates, probably contamination. When plating out the lysates that should be sterile, some bacteria still grew, but they grew much more in the controls. If the contaminated plates are ignored the colicin Fy seems specific, but more experiments need to be done to be sure. Due to lack of time this was not possible.

Figure 6: The E on the plates stands for Enterobacter, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. the numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Figure 7: Controls of the lysate to see if they were contaminated or not, to the left is the control lysate while the bacteriocin is to the right.

As seen in fig. 7 the control lysate is much more contaminated than the bacteriocin, this explains why the control plates in all cases have more bacteria on the plates plated with 300 µl of lysate added compared with all the others. Different types of colonies can also be seen on most of them.
In conclusion there are some indications that colicin Fy is specific but more tests need to be done.

Read more/Hide

Appendix 2: The K on the plates stands for klebsiella, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. the numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Appendix 3: The D on the plates stands for DH5-alpha (E. coli), the C stands for control lysate added while B for bacteriocin (colicin Fy) added. The numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Appendix 4: The P on the plates stands for pseudomonas, the C stands for control lysate added while B for bacteriocin (colicin Fy) added. The numbers 10, 100 and 300 stands for the amount of µl of lysate added.

Parts

Fav.BioBrick codeTypeConstructDescriptionDesigners
BBa_K1381014RegulatoryJ23101-spot42_USP45_10bpSilencing sRNA with affinity to USP45Killing Group
BBa_K1381015RegulatoryJ23101-spot42_USP45_15bpSilencing sRNA with affinity to USP45Killing Group
BBa_K1381016RegulatoryJ23101-spot42_USP45_20bpSilencing sRNA with affinity to USP45Killing Group
BBa_K1381017CodingCFY-X6 hisThe gene coding for the bacteriocin colicin FyKilling Group
BBa_K1381018TagB0034-USP45The secretion tag USP45 with the RBS B0034Killing Group
BBa_K1381019DeviceB0034-pelB-CFY-X6 hisThe bacteriocin colicin Fy coupled to the the secretion tag pelBKilling Group
BBa_K1381020GeneratorJ23106-B0034-pelB-CFY-X6 hisColicin Fy coupled to the the secretion tag pelB with the promoter J23106Killing Group
BBa_K1381021GeneratorJ23116-B0034-pelB-CFY-X6 hisColicin Fy coupled to the the secretion tag pelB with the promoter J23116Killing Group
BBa_K1381022DeviceB0034-USP45-CFY-X6 hisThe bacteriocin colicin Fy coupled to the the secretion tag USP45Killing Group
BBa_K1381023GeneratorJ23106-B0034-CFY-X6 hisColicin Fy coupled to the promoter J23106Killing Group
  • [1] (2013) "Bacteriocins — a viable alternative to antibiotics?" Paul D. Cotter et al
  • [2] (2012) "Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation" J.Bosak et al
  • [3] (2012) https://2012.igem.org/Team:Uppsala_University/Project