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<img src="https://static.igem.org/mediawiki/2017/c/cf/T--TU_Dresden--P_Peptidosomes_at_a_glance.png" class="zoom"></figure> | <img src="https://static.igem.org/mediawiki/2017/c/cf/T--TU_Dresden--P_Peptidosomes_at_a_glance.png" class="zoom"></figure> | ||
<h4>Motivation:</h4> | <h4>Motivation:</h4> | ||
− | <p>Development of a | + | <p>Development of a selectively permeable cage to encapsulate living bacteria with useful properties.</p> |
<p></p> | <p></p> | ||
<h4>Approach:</h4> | <h4>Approach:</h4> | ||
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<p></p> | <p></p> | ||
<h4>Achievements:</h4> | <h4>Achievements:</h4> | ||
− | <p>(I) A robust protocol for | + | <p>(I) A robust protocol for <a href="#creation" class="hashlink">Peptidosome formation</a> was established. (II) Bacteria could be encapsulated and were <a href="#growth" class="hashlink">alive and growing</a> inside the Peptidosomes. (III) <a href="#wellscan" class="hashlink">Reporter strains</a> showed full functionality in Peptidosomes. (IV) Peptidosomes could be functionalized by <a href="#surface" class="hashlink">surface decoration</a>.</p> |
</figure> | </figure> | ||
</div> | </div> | ||
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<h1 class="box-heading">Short Description</h1> | <h1 class="box-heading">Short Description</h1> | ||
<p>Peptidosomes are the new fundamental approach for generating and utilizing encapsulated bacteria. By the creation of spherical compartments containing a liquid environment inside, bacteria are still able to grow and fulfill a given task. The mesh-like structure of the sphere allows the selective exchange of compounds via diffusion, but holds the bacteria trapped inside. Therefore, we are able to benefit from the entrapped cells’ abilities, while still ensuring they are not released into their surroundings. | <p>Peptidosomes are the new fundamental approach for generating and utilizing encapsulated bacteria. By the creation of spherical compartments containing a liquid environment inside, bacteria are still able to grow and fulfill a given task. The mesh-like structure of the sphere allows the selective exchange of compounds via diffusion, but holds the bacteria trapped inside. Therefore, we are able to benefit from the entrapped cells’ abilities, while still ensuring they are not released into their surroundings. | ||
− | Peptidosomes can be further enhanced by | + | Peptidosomes can be further be enhanced by the incorporation of magnetic or biological beads – which themselves can be functionalized with proteins – into their peptide-based fibrillary shell. |
</p> | </p> | ||
</div> | </div> | ||
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Over the last decades, the main focus to increase yields has been on extensive metabolic engineering and optimizing growth conditions. </p> | Over the last decades, the main focus to increase yields has been on extensive metabolic engineering and optimizing growth conditions. </p> | ||
− | <p>Yet, there are more aspects which need to be considered when producing a compound of interest. First, where can the product of interest be found | + | <p>Yet, there are more aspects which need to be considered when producing a compound of interest. First, where can the product of interest be found; inside of the producing strain or will it be secreted to the surrounding media? Second, what is necessary to separate the valuable end-product from the bacteria? And maybe most importantly, how to assure a safe use of genetically engineered production strains? If you would like to know more about encapsulated bacteria in Peptdisomes and boosting the production of a compound of interest check out our: <a href="https://2017.igem.org/Team:TU_Dresden/Measurement">Signal Peptide Toolbox </a> and <a href="https://2017.igem.org/Team:TU_Dresden/Project/Secretion">Peptide Secretion</a> sections. <p> |
− | <p>To address these major biological and technical questions, the TU Dresden iGEM team presents EncaBcillus. Using <i>Bacillus subtilis</i> as model organism we introduce a new fundamental approach for cultivation of bacteria: Peptidsomes. These Peptidosmes are | + | <p>To address these major biological and technical questions, the TU Dresden iGEM team presents EncaBcillus. Using <i>Bacillus subtilis</i> as a model organism we introduce a new fundamental approach for the cultivation of bacteria: Peptidsomes. These Peptidosmes are built up of self-assembled Fmoc dipeptide phenylalanines (Fmoc-FF) and they are able to form spherical cages. The cages encapsulate the bacteria and prevent them from escaping into the surroundings, but the Peptidosomes are freely diffusible for smaller molecules. We show, that <i>B. subtilis</i> is able to grow, while entrapped inside of the Peptidosomes and additionally, we demonstrate the applicability of two (fluorescence and luminescence) reporters for the use in conjunction with Peptidosomes.</p> |
<p>While evaluating this new method of bacterial entrapment, we established two applications using Peptidsomes:</p> | <p>While evaluating this new method of bacterial entrapment, we established two applications using Peptidsomes:</p> | ||
<ul> | <ul> | ||
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<figcaption><b>Figure 1: Chemical structure of Fmoc-FF</b> </figcaption> | <figcaption><b>Figure 1: Chemical structure of Fmoc-FF</b> </figcaption> | ||
</figure> | </figure> | ||
− | <p>Nanostructures are gaining great attention due to their | + | <p>Nanostructures are gaining great attention due to their vast instrumentalities. In general, nanostructure clusters are ordered within nanoscopic dimensions. In some cases, the building blocks of the nanostructures (which can be of organic or inorganic sources) can self-assemble. This process can be driven by non-covalent forces (e.g. hydrogen bonds, van der waals forces or aromatic interactions) that dictate the organization into supramolecular structures <a target="_blank" href="http://pubs.rsc.org/en/content/articlehtml/2016/cs/c5cs00889a">[1]</a>. For our project EncaBcillus, we investigated the encapsulation of bacteria by the self-assembling building block <b>Fmoc-FF</b> (9-fluorenylmethoxycarbonyl diphenylalanine) (Figure 1). </p> |
<p>While studying the mechanisms that direct the self-assembly of amyloid fibrils by short aromatic peptides, it has been observed that the dipeptide diphenylalanine (FF), the core recognition motif of the Alzheimer’s β-amyloid polypeptide, self-assembled into nanotubular structures in aqueous solution <a target="_blank" href="http://science.sciencemag.org/content/300/5619/625.full">[2]</a><a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16733570">[3]</a>. Later, during the investigation of the interactions in the process, the chemical group 9-fluorenylmethoxycarbonyl (Fmoc) was added to the N-terminus of the dipeptide, facilitating the self-assembly into typical amyloid-like fibrils <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1560/5MC0-V3DX-KE0B-YF3J/full">[4]</a>.</p> | <p>While studying the mechanisms that direct the self-assembly of amyloid fibrils by short aromatic peptides, it has been observed that the dipeptide diphenylalanine (FF), the core recognition motif of the Alzheimer’s β-amyloid polypeptide, self-assembled into nanotubular structures in aqueous solution <a target="_blank" href="http://science.sciencemag.org/content/300/5619/625.full">[2]</a><a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16733570">[3]</a>. Later, during the investigation of the interactions in the process, the chemical group 9-fluorenylmethoxycarbonyl (Fmoc) was added to the N-terminus of the dipeptide, facilitating the self-assembly into typical amyloid-like fibrils <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1560/5MC0-V3DX-KE0B-YF3J/full">[4]</a>.</p> | ||
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<figcaption><b>Figure 4: Schematic representation of the encapsulation of bacteria in Peptidosomes.</b> </figcaption> | <figcaption><b>Figure 4: Schematic representation of the encapsulation of bacteria in Peptidosomes.</b> </figcaption> | ||
</figure> | </figure> | ||
− | <p>In EncaBcillus, we characterized functional Peptidosomes encapsulating <i>Bacillus subtilis</i>. This fundamentally new approach of applying Fmoc-FF in combination with bacteria will create endless new possibilities for bacterial | + | <p>In EncaBcillus, we characterized functional Peptidosomes encapsulating <i>Bacillus subtilis</i>. This fundamentally new approach of applying Fmoc-FF in combination with bacteria will create endless new possibilities for bacterial immobilization.</p> |
<p>To encapsulate the bacteria we first needed to establish a lab protocol. We created Fmoc-FF solutions in water, adjusted the pH to 10.5 and resuspended <i>B. subtilis</i> cells either directly taken from overnight cultures or day cultures.</p> | <p>To encapsulate the bacteria we first needed to establish a lab protocol. We created Fmoc-FF solutions in water, adjusted the pH to 10.5 and resuspended <i>B. subtilis</i> cells either directly taken from overnight cultures or day cultures.</p> | ||
<p>To monitor bacterial growth in Peptidosomes, day cultures were grown until an OD<sub>600</sub> of 0.02. Cells were pelleted by centrifugation for 5 min at 16000g. The supernatant was discarded and the pellet was resuspended in Fmoc-FF solution. Droplets of 15 μl of this solution were placed on an ultrahydrophobic membrane and exposed to CO<sub>2</sub>. </p> | <p>To monitor bacterial growth in Peptidosomes, day cultures were grown until an OD<sub>600</sub> of 0.02. Cells were pelleted by centrifugation for 5 min at 16000g. The supernatant was discarded and the pellet was resuspended in Fmoc-FF solution. Droplets of 15 μl of this solution were placed on an ultrahydrophobic membrane and exposed to CO<sub>2</sub>. </p> | ||
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<h3>Characterization of Peptidosomes </h3> | <h3>Characterization of Peptidosomes </h3> | ||
− | <p>Before we could encapsulate bacteria, we had to establish a robust and reproducible protocol of Peptidosome generation under lab conditions. To tackle this task, we used pH indicator cresol red solutions to visualize the generated Peptidosomes (Figure 5). The pH indicator | + | <p>Before we could encapsulate bacteria, we had to establish a robust and reproducible protocol of Peptidosome generation under lab conditions. To tackle this task, we used pH indicator cresol red solutions to visualize the generated Peptidosomes (Figure 5). The pH indicator color also correlated with the status of the Peptidosomes formation. Prior to the CO<sub>2</sub> exposure the indicator was red (Figure 5, A), after sufficient CO<sub>2</sub> exposure (will be discussed later) the solution changed to yellow (Figure 5, B), indicating a self-assembled Fmoc-FF layer and thus ready-to-use Peptidosomes.</p> |
<p>These generated Peptidosomes stayed stable after CO<sub>2</sub> treatment and we were able to transfer them into liquid filled petri-dishes or well plates.</p> | <p>These generated Peptidosomes stayed stable after CO<sub>2</sub> treatment and we were able to transfer them into liquid filled petri-dishes or well plates.</p> | ||
<p>After we have established the protocol for the Peptidosome creation, we evaluated if we could vary the size of the Peptidosomes. For testing this, we simply generated Peptidosomes of different volumes and could demonstrate successful created and stable Peptidosomes ranging from 1 to 20 µl (Figure 6).</p> | <p>After we have established the protocol for the Peptidosome creation, we evaluated if we could vary the size of the Peptidosomes. For testing this, we simply generated Peptidosomes of different volumes and could demonstrate successful created and stable Peptidosomes ranging from 1 to 20 µl (Figure 6).</p> | ||
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</figure> | </figure> | ||
− | <p>Proving that diffusion between the inside of the Peptidosome and the surrounding is possible, we next tested if diffusion between two Peptidosomes which are in direct contact is possible. For this experiment only one of the two connected Peptidosomes contained the | + | <p>Proving that diffusion between the inside of the Peptidosome and the surrounding is possible, we next tested if diffusion between two Peptidosomes which are in direct contact is possible. For this experiment, only one of the two connected Peptidosomes contained the colored pH indicator solution. Over the lapse of 1.5 hours, it was possible to observe a diffusion of the color between both Peptidosomes until it reached an equilibrium (Figure 9). This proved that there is intercommunication, between the two Peptidosomes. </p> |
<p> </p> | <p> </p> | ||
<figure class="makeresponsive" style="width: 65%; display: flex; align-items: center; justify-content: center; flex-direction: column;""> | <figure class="makeresponsive" style="width: 65%; display: flex; align-items: center; justify-content: center; flex-direction: column;""> | ||
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</figure> | </figure> | ||
<p> </p> | <p> </p> | ||
− | <p>Additionally, to the previous described method of Peptidosome generation, we also tried a microinjection-technique. By doing so, we injected a coloured solution to the inside of the Peptidosome (Figure 10). To close the membrane hole that was introduced by the thin glass capillary, the Peptidosome was after injection again exposed to CO<sub>2</sub> for 5 min. Followed by this procedure, the Peptidosome was transferred to water and as shown in Figure 10 E) the | + | <p>Additionally, to the previous described method of Peptidosome generation, we also tried a microinjection-technique. By doing so, we injected a coloured solution to the inside of the Peptidosome (Figure 10). To close the membrane hole that was introduced by the thin glass capillary, the Peptidosome was after injection again exposed to CO<sub>2</sub> for 5 min. Followed by this procedure, the Peptidosome was transferred to water and as shown in Figure 10 E) the Peptidosome was stable. </p> |
<figure class="makeresponsive" style="width: 60%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | <figure class="makeresponsive" style="width: 60%; display: flex; align-items: center; justify-content: center; flex-direction: column;"> | ||
<img src="https://static.igem.org/mediawiki/2017/b/be/T--TU_Dresden--microinjection.png" | <img src="https://static.igem.org/mediawiki/2017/b/be/T--TU_Dresden--microinjection.png" | ||
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<br> | <br> | ||
<h4> Can Fmoc-FF use <i><b>B. subtilis</b></i> as a nitrogen source?</h4> | <h4> Can Fmoc-FF use <i><b>B. subtilis</b></i> as a nitrogen source?</h4> | ||
− | <p>To test | + | <p>To test if <i><b>B. subtilis</b></i> can degrade Fmoc-FF, we tested bacterial growth on Jensen’s medium agar plates. This medium has all the basic nutrients that bacteria needs for growth, with exception of nitrogen.</p> |
<p>The experimentally used agar plates were set-up as followed: </p> | <p>The experimentally used agar plates were set-up as followed: </p> | ||
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<li> Jensen’s medium supplemented with Fmoc-FF after the plates were dried</li> | <li> Jensen’s medium supplemented with Fmoc-FF after the plates were dried</li> | ||
</ol> | </ol> | ||
− | <p>Type one plates serve as negative control, since <i>B. subtilis</i> (wild type) should not be | + | <p>Type one plates serve as negative control, since <i>B. subtilis</i> (wild type) should not be able to grow in the absence of nitrogen. On the second plates, we added two nitrogen sources (Fe-[III]-ammonium citrate and potassium glutamate) allowing <i>B. subtilis</i> to grow. With plates of type three and four we intended to test if <i>B. subtilis</i> is capable of using the Fmoc-FF. We could only detect growth on type two plates, demonstrating that <i>B. subtilis</i> can neither grow with our provided nitrogen (type 1 plates) nor use Fmoc-FF as nitrogen source (type three and four plates). |
</p> | </p> | ||
<p></p> | <p></p> | ||
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<h4><i><b>B. subtilis</b></i> in Peptidosomes</h4> | <h4><i><b>B. subtilis</b></i> in Peptidosomes</h4> | ||
− | <p>After we have proven that <i>B. subtilis</i> can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria started. </p> | + | <p>After we have proven that <i>B. subtilis</i> can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria were started. </p> |
<p>For this we resuspended an appropriate amount of bacteria in the Fmoc-FF solution. Afterwards droplets of this were placed on an ultrahydrophobic membrane and exposed to CO<sub>2</sub> for 10 minutes, followed by a transfer to water or LB medium. </p> | <p>For this we resuspended an appropriate amount of bacteria in the Fmoc-FF solution. Afterwards droplets of this were placed on an ultrahydrophobic membrane and exposed to CO<sub>2</sub> for 10 minutes, followed by a transfer to water or LB medium. </p> | ||
<p>You can find the detailed protocols <a href="https://2017.igem.org/Team:TU_Dresden/Experiments">here</a>.</p> | <p>You can find the detailed protocols <a href="https://2017.igem.org/Team:TU_Dresden/Experiments">here</a>.</p> | ||
<p></p> | <p></p> | ||
− | <h4> Well Scan experiment using <i><b>B. subtilis</b></i> reporter strains </h4> | + | <h4 id="wellscan"> Well Scan experiment using <i><b>B. subtilis</b></i> reporter strains </h4> |
− | <p>For the first experiments of encapsulation, we used the two <i>B. subtilis</i> reporter strains: TMB4131 W168 <i>lacA</i>::<i>erm</i> P<i><sub>veg</sub></i>-<i>sfGFP</i> and TMB3090 W168 <i>sacA</i>::<i>cat</i> P<i><sub>veg</sub></i>-<i>luxABCDE</i>. The first strain constitutively expresses sfGFP, which we used to detect the presence of bacteria in the Peptidosomes by following the fluorescence signal. The latter strain | + | <p>For the first experiments of encapsulation, we used the two <i>B. subtilis</i> reporter strains: TMB4131 W168 <i>lacA</i>::<i>erm</i> P<i><sub>veg</sub></i>-<i>sfGFP</i> and TMB3090 W168 <i>sacA</i>::<i>cat</i> P<i><sub>veg</sub></i>-<i>luxABCDE</i>. The first strain constitutively expresses sfGFP, which we used to detect the presence of bacteria in the Peptidosomes by following the fluorescence signal. The latter strain expresses luciferase in a constitutive way, which makes a detection of a luminescence signal possible. Having both of these well-evaluated readouts at hand, we wanted to demonstrate their applicability, with bacteria encapsulated in Peptidosomes. |
We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green color, while the position of the fluorescence/luminescence source appears as red. Please check out the according <a href="https://2017.igem.org/Team:TU_Dresden/Experiments">protocol</a> for details.</p> | We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green color, while the position of the fluorescence/luminescence source appears as red. Please check out the according <a href="https://2017.igem.org/Team:TU_Dresden/Experiments">protocol</a> for details.</p> | ||
<p>In Figure 12 examples of the well scans of different samples are displayed.</p> | <p>In Figure 12 examples of the well scans of different samples are displayed.</p> | ||
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<h4 id="microscopy"> Fluorescence microscopy with <i><b>B. subtilis</b></i></h4> | <h4 id="microscopy"> Fluorescence microscopy with <i><b>B. subtilis</b></i></h4> | ||
<figure> | <figure> | ||
− | <figure class="makeresponsive | + | <figure class="makeresponsive floatright" style="width: 30%;"> |
<img src="https://static.igem.org/mediawiki/2017/c/ca/T--TU_Dresden--stereo.png" | <img src="https://static.igem.org/mediawiki/2017/c/ca/T--TU_Dresden--stereo.png" | ||
alt="Peptidosome with encapsulated fluorescent bacteria" | alt="Peptidosome with encapsulated fluorescent bacteria" | ||
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− | < | + | <h4 id="growth"> The Growth of <i><b>B. subtilis</b></i> in Peptidosomes</h4> |
− | <p>After we checked the functionality of reporters in Peptidosomes, we were also interested to observe growth of <i>B.subtilis</i> while encapsulated. Since the Peptidosomes should be liquid-filled and allow the exchange of molecules, <i>B.subtilis</i> should be able to grow. To demonstrate this, we loaded Peptidosome with a known amount of bacteria and plated them on agar plates after different time periods of incubation at 37°C. These plates were then incubated | + | <p>After we checked the functionality of reporters in Peptidosomes, we were also interested to observe growth of <i>B. subtilis</i> while encapsulated. Since the Peptidosomes should be liquid-filled and allow the exchange of molecules, <i>B. subtilis</i> should be able to grow. To demonstrate this, we loaded Peptidosome with a known amount of bacteria and plated them on agar plates after different time periods of incubation at 37°C. These plates were then incubated overnight and we documented the colony numbers on the next day (Table 1). We expected to observe an increase of the colony forming units in correlation of longer incubation times. Peptidosomes plated after 0 hours represent the starting bacterial concentration, as these Peptidosomes were plated on agar plates immediately after their generation.</p> |
<p></p> | <p></p> | ||
<figure class="makeresponsive" style="width: 60%; display: flex; align-items: center; justify-content: center; flex-direction: column;""> | <figure class="makeresponsive" style="width: 60%; display: flex; align-items: center; justify-content: center; flex-direction: column;""> | ||
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</figure> | </figure> | ||
<p></p> | <p></p> | ||
− | <p>As observed on the results, the number of colonies in the plates increased after 3.5 h of incubation, when compared to the starting bacterial concentration (0 hours). After 7 hours it was not possible to count the amount of colonies, cause <i>B.subtilis</i> formed a lawn. This clearly showed that <i>B.subtilis</i> was fully capable to grow inside of the Peptidosome cages. Thus, Peptidosomes are indeed suitable for the establishment of bacterial cultures. </p> | + | <p>As observed on the results, the number of colonies in the plates increased after 3.5 h of incubation, when compared to the starting bacterial concentration (0 hours). After 7 hours it was not possible to count the amount of colonies, cause <i>B. subtilis</i> formed a lawn. This clearly showed that <i>B. subtilis</i> was fully capable to grow inside of the Peptidosome cages. Thus, Peptidosomes are indeed suitable for the establishment of bacterial cultures. </p> |
<p></p> | <p></p> | ||
− | < | + | <h4 id="sem">Cryo-Scanning Electron Microscopy</h4> |
− | <p>To fully investigate how <i>B.subtilis</i> is encapsulated in the Peptidosomes, we performed Cryo-Scanning Electron Microscopy ( | + | <p>To fully investigate how <i>B. subtilis</i> is encapsulated in the Peptidosomes, we performed Cryo-Scanning Electron Microscopy (Cryo-SEM). By doing so, we should get inside if the surface of the Peptidosome is also covered by bacterial cells.</p> |
<p> </p> | <p> </p> | ||
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</figure> | </figure> | ||
<p></p> | <p></p> | ||
− | <p>By introducing the treatments, the number of colonies was dramatically reduced from several hundreds to an average of 0.7 for the washing steps and 1.7 for the low pH treatment (Table 2). From here, we concluded that the washing steps are important to reduce the number of cells released in the supernatant, whereas the treatment with acid broth does not add a significant effect on top of this. By introducing the washing steps and also reducing the concentration of cells in the Peptidosomes, the amount of bacteria present on the outer surface of the membrane was dramatically decreased. We also confirmed this by performing a second round of cryo-SEM, | + | <p>By introducing the treatments, the number of colonies was dramatically reduced from several hundreds to an average of 0.7 for the washing steps and 1.7 for the low pH treatment (Table 2). From here, we concluded that the washing steps are important to reduce the number of cells released in the supernatant, whereas the treatment with acid broth does not add a significant effect on top of this. By introducing the washing steps and also reducing the concentration of cells in the Peptidosomes, the amount of bacteria present on the outer surface of the membrane was dramatically decreased. We also confirmed this by performing a second round of cryo-SEM, shown below. </p> |
<p></p> | <p></p> | ||
<figure> | <figure> | ||
− | <figure> | + | <figure style="padding-left: 25%;"> |
<img src="https://static.igem.org/mediawiki/2017/f/f0/T--TU_Dresden--sem2A.png" | <img src="https://static.igem.org/mediawiki/2017/f/f0/T--TU_Dresden--sem2A.png" | ||
alt="A" | alt="A" | ||
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</figure> | </figure> | ||
− | <figure> | + | <figure style="padding-left: 25%;"> |
<img src="https://static.igem.org/mediawiki/2017/f/fc/T--TU_Dresden--sem2C.png" | <img src="https://static.igem.org/mediawiki/2017/f/fc/T--TU_Dresden--sem2C.png" | ||
alt="C" | alt="C" | ||
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</figure> | </figure> | ||
<p></p> | <p></p> | ||
− | <p>In this second round of cryo-SEM the number of detectable bacteria that lie or are integrated | + | <p>In this second round of cryo-SEM the number of detectable bacteria that lie on or are integrated into the membrane is significantly lower. Only a few individual cells are visible and the surface is less wrinkled. (Figure 15 A), B)) </p> |
<p>Only on the Peptidosome with the low pH treatment, a cell that was not integrated in the membrane was observed (Figure 15, D). </p> | <p>Only on the Peptidosome with the low pH treatment, a cell that was not integrated in the membrane was observed (Figure 15, D). </p> | ||
<p>The results of the growth experiment with the additional treatments were confirmed microscopically. There was no significant improvement in the Peptidosomes treated with acid LB medium compared to the sole integration of washing steps. This confirms the reduced number of colonies in the supernatant of the growth experiment when carrying out the additional treatment methods (Table 2 and Figure 15). </p> | <p>The results of the growth experiment with the additional treatments were confirmed microscopically. There was no significant improvement in the Peptidosomes treated with acid LB medium compared to the sole integration of washing steps. This confirms the reduced number of colonies in the supernatant of the growth experiment when carrying out the additional treatment methods (Table 2 and Figure 15). </p> | ||
− | <p>The one loosely attached cell might be a problem for some applications where the surrounding medium is expected to be free of bacteria, therefore, the manufacturing technique of Peptidosomes should be adapted accordingly. An adequate method for this could be introducing the cells by microinjection | + | <p>The one loosely attached cell might be a problem for some applications where the surrounding medium is expected to be free of bacteria, therefore, the manufacturing technique of Peptidosomes should be adapted accordingly. An adequate method for this could be introducing the cells by microinjection since there is not a chance that bacteria can be attached to the outer part of the membrane. </p> |
<hr> | <hr> | ||
<h2 id="surface"> 3. Surface Decoration </h2> | <h2 id="surface"> 3. Surface Decoration </h2> | ||
− | <p>The properties of self-assembled Fmoc-FF | + | <p>The properties of self-assembled Fmoc-FF surfaces allow tiny objects, such as Dynabeads, to be incorporated into the shell of Peptidosomes. Incorporating Dynabeads enables the control of the Peptidosomes' movements in a magnetic field, thereby providing a powerful delivery tool, which can be used in various applications. </p> |
<p></p> | <p></p> | ||
<video src="https://static.igem.org/mediawiki/2017/2/2c/T--TU_Dresden--magneticpeptidosome.mp4" controls class="makeresponsive" style="padding-left: 10%; padding-right: 10%; width: 90%;" poster= "https://static.igem.org/mediawiki/2017/9/99/T--TU_Dresden--magnetosomevideo.png"> | <video src="https://static.igem.org/mediawiki/2017/2/2c/T--TU_Dresden--magneticpeptidosome.mp4" controls class="makeresponsive" style="padding-left: 10%; padding-right: 10%; width: 90%;" poster= "https://static.igem.org/mediawiki/2017/9/99/T--TU_Dresden--magnetosomevideo.png"> | ||
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<div style="display: flex; align-items: flex-start; flex-wrap: wrap;"> | <div style="display: flex; align-items: flex-start; flex-wrap: wrap;"> | ||
<div class="makeresponsive" style="width: 65%;"> | <div class="makeresponsive" style="width: 65%;"> | ||
− | <p>Moreover, the surface of Dynabeads can be decorated with | + | <p>Moreover, the surface of Dynabeads themselves can be decorated with proteins. His-tagged GFP was selected for this decoration allowing an easy imaging procedure. We optimized the Invitrogen protocol to fit our needs and tested the incorporation of decorated Dynabeads with His-tagged GFP into the peptidosome surface in binding/wash buffer and LB media (Figures 16, 17). </p> |
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<p></p> | <p></p> | ||
− | <p> | + | <p>With this, we clearly demonstrated the possibility of Peptidosome surface decoration. Hereby, the GFP serves as a proof of principle that can be exchanged with any other protein of interest and thus can be used for various applications where enzymatic activity of the surface of Peptidosomes or Peptidosome immobilization is required. The surface decoration protocol is compatible with almost any goals due to its flexibility, as the decoration procedure can be performed before and after Peptidosome generation.</p> |
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<h2>Conclusion</h2> | <h2>Conclusion</h2> | ||
− | <p class="survey-quote">Our team successfully | + | <p class="survey-quote">Our team successfully introduced a new immobilization system; the Peptidosome. We established a robust protocol for creating the Peptidosomes and so we are able to encapsulate bacteria that can live and grow inside the Peptidosome. In addition, we managed to functionalize the cages by decorating the surface. We can proudly announce that the Peptidosomes are a new fundamental approach for generating and applying encapsulated bacteria.</p> |
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Latest revision as of 02:13, 2 November 2017