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<h1 class="box-heading">Peptidosomes</h1> | <h1 class="box-heading">Peptidosomes</h1> | ||
<h2>Short description</h2> | <h2>Short description</h2> | ||
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<h2> Achievements</h2> | <h2> Achievements</h2> | ||
<p> | <p> | ||
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<h2>Short description</h2> | <h2>Short description</h2> | ||
<p>In bacteria, protein secretion is mainly orchestrated by the Sec Pathway via Signal Peptides (SP), which are located at the N-terminus of secreted proteins. The secretion efficiency is not determined by the sequence of the SP alone, but instead is the combined result of an SP with its specific target protein. This necessitates establishing efficient screening procedures to evaluate all possible SP/target protein combinations. We developed such an approach for our Signal Peptide Toolbox, which contains 74 Sec-dependent SPs. It combines combinatorial construction with highly reproducible, quantitative measurements. By applying this procedure, we demonstrate the secretion of three different proteins and succeeded in identifying the most potent SP-protein combination for each of them. This thoroughly evaluated measurement tool, in combination with our SP toolbox (fully available via the partsregistry) enables an organism-independent, straightforward approach to identifying the best combination of SP with any protein of interest.</p> | <p>In bacteria, protein secretion is mainly orchestrated by the Sec Pathway via Signal Peptides (SP), which are located at the N-terminus of secreted proteins. The secretion efficiency is not determined by the sequence of the SP alone, but instead is the combined result of an SP with its specific target protein. This necessitates establishing efficient screening procedures to evaluate all possible SP/target protein combinations. We developed such an approach for our Signal Peptide Toolbox, which contains 74 Sec-dependent SPs. It combines combinatorial construction with highly reproducible, quantitative measurements. By applying this procedure, we demonstrate the secretion of three different proteins and succeeded in identifying the most potent SP-protein combination for each of them. This thoroughly evaluated measurement tool, in combination with our SP toolbox (fully available via the partsregistry) enables an organism-independent, straightforward approach to identifying the best combination of SP with any protein of interest.</p> | ||
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<h2>Achievements</h2> | <h2>Achievements</h2> | ||
</div> | </div> | ||
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<h2>Short description</h2> | <h2>Short description</h2> | ||
<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> | <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> | ||
+ | <hr> | ||
<h2>Achievements</h2> | <h2>Achievements</h2> | ||
</div> | </div> | ||
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<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> | <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> | ||
+ | <hr> | ||
<p> We were able to engineer <i> B. subtilis </i> to secret large quantities of mCherry constructs, c-terminally fused with a mini. SpyCatcher or SpyTag (Tags). In Figure 1 we assayed the fluorescence in the supernatant, that surpasses the wilde type by far. The typical red color of mCherry is even visible in the supernatant under day light conditions (Figure 2). </p> | <p> We were able to engineer <i> B. subtilis </i> to secret large quantities of mCherry constructs, c-terminally fused with a mini. SpyCatcher or SpyTag (Tags). In Figure 1 we assayed the fluorescence in the supernatant, that surpasses the wilde type by far. The typical red color of mCherry is even visible in the supernatant under day light conditions (Figure 2). </p> | ||
Revision as of 23:50, 29 October 2017