alt="Figure 2: Supernatants of <i>B. subtilis</i> cultures."
alt="Figure 2: Supernatants of <i>B. subtilis</i> cultures."
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<figcaption><b>Figure 2: Supernatants of <i>B. subtilis </i>cultures.</b>
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<figcaption><b>Figure 2: Supernatants of <i><b>B. subtilis</b> </i>cultures.</b>
Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation.
Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation.
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Revision as of 20:20, 28 October 2017
Mission Accomplished
Signal Peptide Toolbox
Short description
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.
Achievements
Evaluation Vector
Short description
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.
Achievements
Secretion
Short description
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.
Achievements
We were able to
Communication
Short description
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.