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<h1 class="box-heading"> Evaluation Vector</h1> | <h1 class="box-heading"> Evaluation Vector</h1> | ||
<h2>Short description</h2> | <h2>Short description</h2> | ||
− | <p>Peptidosomes in combination with Bacillus subtilis offer a perfect platform for enhanced protein overproduction by the means of efficient protein secretion provided through B. subtilis and the easy purification due to the physical separation of bacteria and the end-product in the supernatant facilitated by the Peptidosomes. Naturally, B. subtilis is a strong secretion host and in order to take full advantage of this great potential it is necessary to evaluate all possible combinations of the B. subtilis’ secretion signal peptides and the proteins of interest. Therefore, we developed the Evaluation Vector (EV) which is a powerful genetic tool containing a multiple cloning site (MCS) specifically designed to easily exchange translational fusions composed of the desired protein and a secretion signal peptide.</p> | + | <p>Peptidosomes in combination with <i>Bacillus subtilis</i> offer a perfect platform for enhanced protein overproduction by the means of efficient protein secretion provided through <i>B. subtilis</i> and the easy purification due to the physical separation of bacteria and the end-product in the supernatant facilitated by the Peptidosomes. Naturally, <i>B. subtilis</i> is a strong secretion host and in order to take full advantage of this great potential it is necessary to evaluate all possible combinations of the <i>B. subtilis’</i> secretion signal peptides and the proteins of interest. Therefore, we developed the Evaluation Vector (EV) which is a powerful genetic tool containing a multiple cloning site (MCS) specifically designed to easily exchange translational fusions composed of the desired protein and a secretion signal peptide.</p> |
<h2>Achievements</h2> | <h2>Achievements</h2> | ||
</div> | </div> | ||
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<h1 class="box-heading"> Secretion</h1> | <h1 class="box-heading"> Secretion</h1> | ||
<h2>Short description</h2> | <h2>Short description</h2> | ||
− | <p>In combing Bacillus subtilis powerful secretion capacity with Peptidosomes as a new platform for functional co-cultivation we aim to produce multi protein complexes. Various strains - each secreting distinct proteins of interest - can be cultivated in one reaction hub while being physically separated. In this part of EncaBcillus we study extracelluar protein interaction mediated by the SpyTag/SpyCatcher system. This set-up bears the potential for an effective production of customizable biomaterials or enzyme complexes.</p> | + | <p>In combing <i>Bacillus subtilis</i> powerful secretion capacity with Peptidosomes as a new platform for functional co-cultivation we aim to produce multi protein complexes. Various strains - each secreting distinct proteins of interest - can be cultivated in one reaction hub while being physically separated. In this part of EncaBcillus we study extracelluar protein interaction mediated by the SpyTag/SpyCatcher system. This set-up bears the potential for an effective production of customizable biomaterials or enzyme complexes.</p> |
<h2>Achievements</h2> | <h2>Achievements</h2> | ||
<p> We were able to | <p> We were able to | ||
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<img src="https://static.igem.org/mediawiki/2017/b/bb/T--TU_Dresden--secretion--results7.png" | <img src="https://static.igem.org/mediawiki/2017/b/bb/T--TU_Dresden--secretion--results7.png" | ||
alt="Figure 3: SDS gel with crude and purified supernatants. " class="zoom"> | alt="Figure 3: SDS gel with crude and purified supernatants. " class="zoom"> | ||
− | <figcaption><b>Figure 3: SDS gel with crude and purified supernatants.</b> Expression of the multi copy mCherry constructs was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. The his-tagged proteins were purified with Ni-NTA agarose beads. Lane 1 was loaded with 3 l of NEB´s “Color Prestained Protein Standard Broad Range” ladder. Crude (c) and purified (p) supernatant of wild-type (WT) are shown as a control in lane 2 and 3. Lane 4 and 5 contain the supernatant of B. subtilis producing mCherry-mini. SpyCatcher fusion protein (36,6 kDa). Lane 4 and 5 contain the supernatant of B. subtilis producing mCherry-SpyTag fusion protein (31,9 kDa). The crude supernatants of the two mCherry producing strains were combined, incubated for 4 h, purified and loaded onto lane 8 and 9. The fusion product of the mCherry constructs is visable in the crude and purified supernatant. | + | <figcaption><b>Figure 3: SDS gel with crude and purified supernatants.</b> Expression of the multi copy mCherry constructs was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. The his-tagged proteins were purified with Ni-NTA agarose beads. Lane 1 was loaded with 3 l of NEB´s “Color Prestained Protein Standard Broad Range” ladder. Crude (c) and purified (p) supernatant of wild-type (WT) are shown as a control in lane 2 and 3. Lane 4 and 5 contain the supernatant of <i>B. subtilis</i> producing mCherry-mini. SpyCatcher fusion protein (36,6 kDa). Lane 4 and 5 contain the supernatant of <i>B. subtilis</i> producing mCherry-SpyTag fusion protein (31,9 kDa). The crude supernatants of the two mCherry producing strains were combined, incubated for 4 h, purified and loaded onto lane 8 and 9. The fusion product of the mCherry constructs is visable in the crude and purified supernatant. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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<h1 class="box-heading"> Communication</h1> | <h1 class="box-heading"> Communication</h1> | ||
<h2>Short description</h2> | <h2>Short description</h2> | ||
− | <p>Peptidosomes can be a powerful co-culture technique to physically separate bacterial populations without limiting their ability to communicate with each other by exchanging signalling molecules. This part of EncaBcillus is focused on proofing the concept of communication between encapsulated bacteria by using the regulatory system for competence development in Bacillus subtilis which is based on quorum sensing.</p> | + | <p>Peptidosomes can be a powerful co-culture technique to physically separate bacterial populations without limiting their ability to communicate with each other by exchanging signalling molecules. This part of EncaBcillus is focused on proofing the concept of communication between encapsulated bacteria by using the regulatory system for competence development in <i>Bacillus subtilis</i> which is based on quorum sensing.</p> |
<h2>Achievements</h2> | <h2>Achievements</h2> | ||
</div> | </div> |
Revision as of 19:40, 28 October 2017