Difference between revisions of "Team:TU Dresden/Demonstrate"

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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>
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<p> We were able to  
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<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>
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<p>The functionality of the fused Tags could be proven with an SDS-PAGE (Figure 3). Upon 4 h of incubating the supernatants containing mCherry with either SpyTag or mini. SpyCatcher, the conjugated fusion product could be detected. This proves the functionality of our fusion constructs, opening possibilities for co-culture approaches using Peptidosomes, to produce self conjugation protein complexes. <p>
 
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Revision as of 13:08, 29 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 engineer B. subtilis 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).

The functionality of the fused Tags could be proven with an SDS-PAGE (Figure 3). Upon 4 h of incubating the supernatants containing mCherry with either SpyTag or mini. SpyCatcher, the conjugated fusion product could be detected. This proves the functionality of our fusion constructs, opening possibilities for co-culture approaches using Peptidosomes, to produce self conjugation protein complexes.

Figure 1: Endpoint measurement of the fluorescence from supernatants.
Figure 1: Endpoint measurement of the fluorescence from supernatants. Expression of the multi copy mCherry constructs (purple) was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength was set to 585 nm and emission was recorded at 615 nm. The fluorecense was normalized over the optical density of the cell culture at 600 nm (OD600). Graph shows mean values and standard deviations of at least two biological and three technical replicates.
Figure 2: Supernatants of <i>B. subtilis</i> cultures.
Figure 2: Supernatants of B. subtilis cultures. 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.
Figure 3: SDS gel with crude and purified supernatants.
Figure 3: SDS gel with crude and purified supernatants. 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.

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.

Achievements