Team:Uppsala/Project Adhesion

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">In our project we developed a system to fight pathogens in the gut, which is not a unique idea. Except we wanted our systems key attribute to be specificity. To do so, we needed to find a unique quorum sensing language that we could hijack and utilize. We found the Yen system, from <i>Yersinia enterocolitica</i>. A homologous sytem to the well known Lux system, from <i>Vibrio fischeri</i>.</p>
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<p class="box_text">To maximize the specificity in targeting <i>Yersinia enterocolitica</i>, we wanted to design a construct consisting of a membrane protein complex that would bind to the surface of the pathogen. This could be done by fusing an anchor protein with the colicin Fy that aimed at the membrane of <i>Y. enterocolitica</i>. This complex would be expressed on the surface of our probiotic, making it adhere to its target.</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">From the Yen system we stole the activator YenR and the recognition region, the yenbox, fused with a wild type promoter. YenR was synthesized with the RBS B0034 and codon optimized for <i>E. coli</i>. A customized version of the yenbox-promoter was also created.</p>
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<p class="box_text">We made two constructs, using a synthesized gene coding for pgsA. In the first one we fused pgsA with colicin Fy and added a promoter aswell as a RBS called B0034. In the other, BFP was put instead of colicin Fy to test the expression of pgsA.</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 get a five-fold induction relative to the base level of expression. The cells that were used during the measurements were double transformed with the yenbox-promoter complex with GFP and a plasmid producing the activator YenR.</p>
+
<p class="box_text">Mutagenesis of B0034-BFP into RFC25 was successful, verified by sequencing. All other constructs were not successful. Mainly because we had trouble with the RFC25-assemblies. The removal of TACTAG was also not successful.</p>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381000">BBa_K1381000</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381024">BBa_K1381024</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381001">BBa_K1381001</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381025">BBa_K1381025</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381002">BBa_K1381002</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381004">BBa_K1381004</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381005">BBa_K1381005</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381006">BBa_K1381006</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381007">BBa_K1381007</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381008">BBa_K1381008</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381009">BBa_K1381009</a></li>
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<li class="mparts"><a href="http://parts.igem.org/Part:BBa_K1381010">BBa_K1381010</a></li>
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<br><br>
<br><br>
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<a id="ref_point"></a><h2 class="project_h2">Background</h2>
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<a id="ref_point"></a><h2>Background</h2>
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<p> When creating a biological machine that efficiently can kill off specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our probiotic to attach to its target, like Yersinia enterocolitica in our case, could give important advantages in target specificity and delivering medicine in a way that ensures it reaches Y. enterocolitica in a concentration as high as possible. The idea is to manipulate Lactobacillus expression of membrane proteins to create a somewhat stable bonding to any of Yersina’s surface structures, preventing the pathogen from even entering the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that Yersinia makes to our cells or one that itself binds to Yersinia. <br><br> We studied the ways of infection of Yersinia Enterocolitica and could determine that the main strategies of entering through the gut wall is by using it’s diverse membrane proteins YadA, Invasin and Ail. YadA and Invasin work together to bind to our cell membranes. While YadA binds to collagen on our cells, Invasin structurally resembles fibronectin , that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein Integrin alpha-5-beta-1 in our cell mambranes. [1] Yersinia uses our own cell-to-cell binding mechanism to find a target cell and fooling it to get inside by binding to the Integrin and to the collagen outside and in between cells. [2] <br> At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen like proteins by fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen part attached to the probiotic surface. YadA binds strongly to some parts of collagen which could be expressed individually. [3] <br><br> But by experimenting with collagen, and especially putting it on the surface of a bacterial outer membrane that is meant to enter the human body, is an extremely risky procedure. “As anti-bodies would be produced to target the collagen and anchor protein complex this could lead to an auto immune reaction as they would also react on the collagen naturally present in our body” says Lars Hellman, a professor in immunology in Uppsala University. More about Problems with Yersinia adhesion and developing auto immune disease can be read in this text:TEXT link. <br><br> Instead we took inspiration from the killing system we are using which secrets a bacteriocin, CFY, which specifically binds to and ruptures the outer cell membrane of Yersinia.[4] The main focus of the adhesion system drastically changed from expressing human like proteins to expressing the bacteriocine fused with the anchor protein. This process is less studied, as the bacteriocin was first discovered and characterized as early as 2012.[5] If the bacteriocin would survive degradation in stomach like environments and later on find Y. enterocolitica, the bacteriocine, while attaching to the membrane, would somewhat fix the probiotic to its target. Even thou this connection would be much weaker than a system with collagen it is safe and still an advantage to the pathogen killer.</p>
+
<h3>Purpose of making an adhesion system between <i>Y.enterocolitica</i> and the Bactissile</h3>
 +
<p> While creating a biological machine that can efficiently kill specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our Bactissile to attach to its target, in our case <i>Yersinia enterocolitica</i> , could give important advantages in target specificity and delivering medicine in a way that ensures it reaches <i>Y. enterocolitica</i> in a concentration as high as possible.</p>
-
<a id="ref_point2"></a><h2 class="project_h2">System design</h2>
+
<h3>Technical Approach</h3>
-
<p>hen creating a biological machine that efficiently can kill off specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our probiotic to attach to its target, like Yersinia enterocolitica in our case, could give important advantages in target specificity and delivering medicine in a way that ensures it reaches Y. enterocolitica in a concentration as high as possible. The idea is to manipulate Lactobacillus expression of membrane proteins to create a somewhat stable bonding to any of Yersina’s surface structures, preventing the pathogen from even entering the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that Yersinia makes to our cells or one that itself binds to Yersinia. <br><br> We studied the ways of infection of Yersinia Enterocolitica and</p>
+
<p>The idea was to manipulate our Bactissiles bonding to any of <i>Y. enterocoliticas</i> surface structures and thus preventing the pathogen from ever attaching to the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that <i>Y. enterocolitica</i> makes to our cells or one that itself binds to <i>Y. enterocolitica</i>.
 +
<br><br>
 +
 
 +
We studied the ways of infection of <i>Y. enterocolitica</i> and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.<sup><a href="#reference1">[1]</a></sup> <i>Y. enterocolitica</i> uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.<sup><a href="#reference2">[2]</a></sup>
 +
At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.<sup><a href="#reference3">[3]</a></sup>
 +
<br><br>
 +
By using collagen, and especially putting it on the surface of a bacterial outer membrane that is meant to enter the human body, is an extremely risky procedure. <i>“As antibodies would be produced to target the collagen and anchor protein complex this could lead to an autoimmune reaction as they would also react on the collagen naturally present in our body”</i> says Lars Hellman, a professor in immunology at Uppsala University. More about problems with <i>Y. enterocolitica</i>-adhesion and the development of autoimmune  disease can be found in this <a href="https://2014.igem.org/Team:Uppsala/Safety">text</a>.
 +
<br><br>
 +
 
 +
Instead we took inspiration from our teams killing system which secretes a bacteriocin, CFY, which specifically binds to and ruptures the outer cell membrane of <i>Y. enterocolitica</i>.<sup><a href="reference4">[4]</a></sup> The main focus of the adhesion system drastically changed from expressing human like proteins to expressing the bacteriocin fused with the anchor protein.
 +
<br><br>
 +
If the bacteriocin would survive degradation in stomach like environments and later on find <i>Y. enterocolitica</i>, the bacteriocin, while attaching to the membrane, would  fix the probiotic to its target. Even though this connection would be much weaker than a system with collagen, it is safe and still an advantage to the Bactissile.</p>
 +
 
 +
<a id="ref_point3"></a><h2>System Design</h2>
 +
<h3>The Anchor</h3>
 +
<p>Since we want our system to be usable in a probiotic bacteria we looked for an anchor motif that works both in our well characterised prototype bacteria <i>(E. coli)</i> and in a well known probiotic bacteria like <i>Lactobacillus</i>. We also wanted the anchor motif to be usable with a wide range of protein types since this would be a great addition to the iGEM registry, since no construct for cell surface display could be found by us at the start of our project. In the end we found the gene coding for PgsA from <i>Bacillus subtilis</i> that had been used successfully in both <i>E.coli</i> and a <i>Lactobacillus</i> species. To prevent interaction with the fused domain we added a long (20aa) rigid linker [BBa_J176131] to the C-terminal of the anchor. </p>
 +
<h3>The fusion</h3>
 +
<p>
 +
To make the fusion protein of anchor and colicin Fy (PgsA-CFY) we chose to synthesize the genes in RFC25 standard (Freiburg). This adds two additional restriction enzyme sites (NgoMIV and AgeI) to the four standard ones, thus enabling simple assembly of the two proteins so that they are translated as one in the same reading frame.
 +
</p>
 +
<h3>Back up plan</h3>
 +
<p>We used normal assembly (RFC10) with re-ligation of XbaI and SpeI sites that leaves a TACTAG-scar where TAG can stop translation and therefore disable production of a fusion protein. A solution would then be to perform PCR-mutagenesis and remove the TACTAG-scar via point mutation.
 +
</p>
 +
<h3>Characterising the anchor</h3>
 +
<p>To test that the anchor attaches to the membrane, we tried to fuse a reporter, the blue fluorescence protein BFP, to the anchor. We chose to mutagenise B0034-BFP [BBa_K592024] to make it compatible with RFC25. It was designed to get an ATG followed by an NgoMIV site upstream of the BFP CDS. The original ATG from the BFP CDS was removed. In the end we would still have to mutagenise BFP, because we never got the RFC25 assembly to work.
 +
</p>
 +
 
 +
<a id="ref_point2"></a><h2>Results</h2>
 +
<p>Mutagenesis of BFP into RFC25 was successful, verified by sequencing. All other constructs were not successful. Mainly because we had trouble with the RFC25-assemblies. The mutagenesis to remove TACTAG also failed because the reverse primer annealed to a region upstream of the desired site causing a frameshift verified by sequencing. The repeat-like sequence of the 60bp linker was probably one of the reasons for this.
 +
</p>
 +
<a id="ref_point4"></a><h2>Parts</h2>
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<a id="ref_point3"></a><h2 class="project_h2">Result</h2>
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<table id=partsT style="width:100%">
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<p>hen creating a biological machine that efficiently can kill off specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our probiotic to attach to its target, like Yersinia enterocolitica in our case, could give important advantages in target specificity and delivering medicine in a way that ensures it reaches Y. enterocolitica in a concentration as high as possible. The idea is to manipulate Lactobacillus expression of membrane proteins to create a somewhat stable bonding to any of Yersina’s surface structures, preventing the pathogen from even entering the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that Yersinia makes to our cells or one that itself binds to Yersinia. <br><br> We studied the ways of infection of Yersinia Enterocolitica and</p>
+
<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_K1381024">BBa_K1381024</a></td><td>Tag</td><td>B0034-pgsA-(EAAAR)x4</td><td>The anchor tag pgsA with the RBS B0034 and a linker</td><td>Adhesion Group</td></tr>
 +
<tr><td></td><td><a href="http://parts.igem.org/Part:BBa_K1381025">BBa_K1381025</a></td><td>Reporter</td><td>B0034-BFP (RFC25)</td><td>A mutagenised version of B0034-BFP, compatible with RFC25</td><td>Adhesion Group</td></tr>
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</table>
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<ul class="reference"><li>[1] <a href="http://edoc.rki.de/oa/articles/reeQGIo7WUhm/PDF/247mpj5mInEcs.pdf">http://edoc.rki.de/oa/articles/reeQGIo7WUhm/PDF/247mpj5mInEcs.pdf</a></li><li>[2] <a href="http://edoc.rki.de/oa/articles/reeQGIo7WUhm/PDF/247mpj5mInEcs.pdf">http://edoc.rki.de/oa/articles/reeQGIo7WUhm/PDF/247mpj5mInEcs.pdf</a> </li></ul>
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<ul class="reference">
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<li><a id="reference1">[1]</a> (2012) “Bacterial Cell Surface Structures of Yersinia”, N. Bialas</li>
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<li><a id="reference2">[2]</a> (2011) “Unique Cell Adhesion and Invasion Properties of Yersinia enterocolitica” O:3, the Most Frequent Cause of Human Yersiniosis” by F. Piscano et.al.</li>
 +
<li><a id="reference3">[3]</a> (2001) “YadA, the multifaceted Yersinia adhesin” by J. El Tahir, M. Skurnik.</li>
 +
<li><a id="reference4">[4]</a> (2012) “Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation“ by J. Bosak et al. </li>
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Background

Purpose of making an adhesion system between Y.enterocolitica and the Bactissile

While creating a biological machine that can efficiently kill specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our Bactissile to attach to its target, in our case Yersinia enterocolitica , could give important advantages in target specificity and delivering medicine in a way that ensures it reaches Y. enterocolitica in a concentration as high as possible.

Technical Approach

The idea was to manipulate our Bactissiles bonding to any of Y. enterocoliticas surface structures and thus preventing the pathogen from ever attaching to the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that Y. enterocolitica makes to our cells or one that itself binds to Y. enterocolitica.

We studied the ways of infection of Y. enterocolitica and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.[1] Y. enterocolitica uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.[2] At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.[3]

By using collagen, and especially putting it on the surface of a bacterial outer membrane that is meant to enter the human body, is an extremely risky procedure. “As antibodies would be produced to target the collagen and anchor protein complex this could lead to an autoimmune reaction as they would also react on the collagen naturally present in our body” says Lars Hellman, a professor in immunology at Uppsala University. More about problems with Y. enterocolitica-adhesion and the development of autoimmune disease can be found in this text.

Instead we took inspiration from our teams killing system which secretes a bacteriocin, CFY, which specifically binds to and ruptures the outer cell membrane of Y. enterocolitica.[4] The main focus of the adhesion system drastically changed from expressing human like proteins to expressing the bacteriocin fused with the anchor protein.

If the bacteriocin would survive degradation in stomach like environments and later on find Y. enterocolitica, the bacteriocin, while attaching to the membrane, would fix the probiotic to its target. Even though this connection would be much weaker than a system with collagen, it is safe and still an advantage to the Bactissile.

System Design

The Anchor

Since we want our system to be usable in a probiotic bacteria we looked for an anchor motif that works both in our well characterised prototype bacteria (E. coli) and in a well known probiotic bacteria like Lactobacillus. We also wanted the anchor motif to be usable with a wide range of protein types since this would be a great addition to the iGEM registry, since no construct for cell surface display could be found by us at the start of our project. In the end we found the gene coding for PgsA from Bacillus subtilis that had been used successfully in both E.coli and a Lactobacillus species. To prevent interaction with the fused domain we added a long (20aa) rigid linker [BBa_J176131] to the C-terminal of the anchor.

The fusion

To make the fusion protein of anchor and colicin Fy (PgsA-CFY) we chose to synthesize the genes in RFC25 standard (Freiburg). This adds two additional restriction enzyme sites (NgoMIV and AgeI) to the four standard ones, thus enabling simple assembly of the two proteins so that they are translated as one in the same reading frame.

Back up plan

We used normal assembly (RFC10) with re-ligation of XbaI and SpeI sites that leaves a TACTAG-scar where TAG can stop translation and therefore disable production of a fusion protein. A solution would then be to perform PCR-mutagenesis and remove the TACTAG-scar via point mutation.

Characterising the anchor

To test that the anchor attaches to the membrane, we tried to fuse a reporter, the blue fluorescence protein BFP, to the anchor. We chose to mutagenise B0034-BFP [BBa_K592024] to make it compatible with RFC25. It was designed to get an ATG followed by an NgoMIV site upstream of the BFP CDS. The original ATG from the BFP CDS was removed. In the end we would still have to mutagenise BFP, because we never got the RFC25 assembly to work.

Results

Mutagenesis of BFP into RFC25 was successful, verified by sequencing. All other constructs were not successful. Mainly because we had trouble with the RFC25-assemblies. The mutagenesis to remove TACTAG also failed because the reverse primer annealed to a region upstream of the desired site causing a frameshift verified by sequencing. The repeat-like sequence of the 60bp linker was probably one of the reasons for this.

Parts

Fav.BioBrick codeTypeConstructDescriptionDesigners
BBa_K1381024TagB0034-pgsA-(EAAAR)x4The anchor tag pgsA with the RBS B0034 and a linkerAdhesion Group
BBa_K1381025ReporterB0034-BFP (RFC25)A mutagenised version of B0034-BFP, compatible with RFC25Adhesion Group
  • [1] (2012) “Bacterial Cell Surface Structures of Yersinia”, N. Bialas
  • [2] (2011) “Unique Cell Adhesion and Invasion Properties of Yersinia enterocolitica” O:3, the Most Frequent Cause of Human Yersiniosis” by F. Piscano et.al.
  • [3] (2001) “YadA, the multifaceted Yersinia adhesin” by J. El Tahir, M. Skurnik.
  • [4] (2012) “Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation“ by J. Bosak et al.