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<p>We engineered a sender strain (SeSt) with an additional inducible copy of <i>comX</i> and a <i>comX</i>-deficient receiver strain (ReSt) containing the ComX-dependent promoter P<sub><i>srfA</i></sub>  fused to the <i>lux</i> operon(Figure 1). Therefore, we could easily detect communication between the co-cultured SeSt and ReSt via ComX by measuring the luminescence output of the ReSt. After proofing this concept using ThinCert™ cell culture inserts (Figure 2) we applied it to Peptidosomes (Figure 3). Consequently we could show communication between encapsulated bacteria and bacteria in the surroundings and made substantial progress in the evaluation of Peptidosomes as a tool for co-cultivation and studies of microbial interactions.</p>
 
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Revision as of 22:28, 30 October 2017

Mission Accomplished

Peptidosomes

Short description

Achievements

Stability and Diffusion

Figure 1, Figure 2

Encapsulation of bacteria

Figure 3, Figure 4

Surface decoration

Figure 5, Video magnetic

Beta-Lactam Biosensor

Short description

Worldwide, multidrug-resistant bacteria are on the rise and provoke the intensive search for novel effective compounds. To simplify the search for new antibiotics and to track the antibiotic pollution in water samples, whole-cell biosensors constitute a helpful investigative tool. In this part of EncaBcillus, we developed a functional and independent heterologous Beta-lactam biosensor in Bacillus subtilis. These specialised cells are capable of sensing a compound of the beta-lactam family and will respond by the production of an easily measurable luminescence signal. We analysed the detection range and sensitivity of the biosensor in response to six different Beta-lactam antibiotics from various subclasses. The evaluated biosensor was then encapsulated into Peptidosomes to proof the concept of our project EncaBcillus. The encapsulation of engineered bacteria allows an simplified handling and increased biosafety, potentially raising the chances for their application in e.g. sewage treatment plants.

Achievements

In this part of the EncaBcillus project, we successfully created and evaluated a novel completely heterologous biosensor for Beta-lactam antibiotics in Bacillus subtilis. This biosensor is able to detect the following Beta-Lactam antibiotics: ampicillin, carbenicillin, cefperazone, cefalexin. cefoxitin and penicillin G in liquid and on solid MH-Medium as illustrated in Figure 1 and 2. Besides the detection range, we analyzed the sensitivity of the biosensor for these specific compounds in several dose-response experiments shown in Figure 3. Furthermore, we demonstrated the applicability of the biosensor when encapsulated into Peptidosomes. As depicted in Figure 4, the biosensor was able to sense the beta-lactam diffusing through the membrane of the Peptidosome. Hereby, we proved the possibility of encapsulating functional engineered bacteria into Peptidosomes and therefore the concept behind our project EncaBcillus.

Figure 5: Bar Charts showing the detection range of the Biosensors
Figure 5: RLU/OD600 values of the different biosensors and the controls are shown 2 hours after induction with the six beta-lactams, bacitracin and dH2O. Graphs show the Wild-type (black), control 1 (light gray), control 2 (dark gray), biosensor 1 (pink), biosensor 2 (purple), biosensor 3 (white and black) and biosensor 3 Xylose induced (dark blue). Luminescence (RLU/OD600) output is shown two hours after beta-lactam antibiotic induction. Mean values and standard deviation are depicted from at least three biological replicates. (3).
Figure 7: Disk Diffusion Assay showing the Biosensor`s activity on solid agar plates
Figure 7: Photographs of the plates from the disk diffusion assay. The upper row shows a picture of the strains at daylight, while the row beneath shows the plate after detection of chemiluminescence (2 minutes exposure time). At the bottom, the disk layout and the most important remarks of the genotype of all strains are indicated.

Figure 9: Dose-Response Curves of the Biosensor
Figure 9: Dose-Response Curves of the six different beta-Lactam antibiotics of biosensor 2 Observed luminescence signal (two hours after antibiotic exposure) was plotted according to each tested antibiotic concentration. Please note both axes are depicted logarithmic. Mean values and standard deviation are depicted from at least three biological replicates.
Figure 10: Encapsulation of the biosensor into Peptidosomes
Figure 10: Encapsulation Experiment with Biosensor 1.The pictures in the upper row show the distribution of the Peptidosomes at the time point of luminescence detection. The plate layout is defined in the legend beneath the photographs. Pink arrows indicate Peptidosomes with a luminescence signal due to the encapsulated biosensor. Upper row of well plate: non-induced samples. Lower row of well plate: induced with cefoperazone (0.2µg µl-1).

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).

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.

We demonstrated the functionality of our SpyTag/SpyCatcher system via SDS-PAGE (Figure 3). Upon 4 h of incubating the supernatants containing mCherry with either SpyTag or mini. SpyCatcher, we were able to detect the conjugated fusion protein. Thus, we provide evidence for the applicability of co-culturing approaches using Peptidosomes, to produce self conjugation protein complexes.

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

By using Peptidosomes we introduce a new powerful platform for co-culturing. This technique physically separates bacterial populations without limiting their ability to communicate with each other via signalling molecules. This part of EncaBcillus is focused on proofing the concept of communication between encapsulated bacteria by making use of the native regulatory system for competence development in Bacillus subtilis which is based on quorum sensing mediated by the ComX pheromone.

Achievements

We engineered a sender strain (SeSt) with an additional inducible copy of comX and a comX-deficient receiver strain (ReSt) containing the ComX-dependent promoter PsrfA fused to the lux operon(Figure 1). Therefore, we could easily detect communication between the co-cultured SeSt and ReSt via ComX by measuring the luminescence output of the ReSt. After proofing this concept using ThinCert™ cell culture inserts (Figure 2) we applied it to Peptidosomes (Figure 3). Consequently we could show communication between encapsulated bacteria and bacteria in the surroundings and made substantial progress in the evaluation of Peptidosomes as a tool for co-cultivation and studies of microbial interactions.

Figure 1: Growth of the ReSt (A) and ComX-dependent promoter activity of P<sub><i>srfA</i></sub> (B) in transformation medium (MNGE).
Figure 1: Growth of the ReSt (A) and ComX-dependent promoter activity of PsrfA (B) in transformation medium (MNGE). In wild type (WT) the promoter is tenfold increased compared to the comX-deficient strain during transition phase.
Figure 2: Co-culture of sender strain (SeSt) and receiver strain (ReSt) using ThinCert™ cell culture inserts.
Figure 2: Co-culture of sender strain (SeSt) and receiver strain (ReSt) using ThinCert™ cell culture inserts. The increasing luminescence of the ReSt (inside of the well) co-cultured with the SeSt (found in the insert) induced with xylose (SeSt + ReSt) was documented by a chemoluminescence imaging system. Cultures containing only SeSt or ReSt served as negative controls. The latter exhibites a weak increase of luminescence because of the promoter’s basal activity (t5).
Figure 3: Co-culture of sender strain (SeSt) and receiver strain (ReSt) using Peptidosomes.
Figure 3: Co-culture of sender strain (SeSt) and receiver strain (ReSt) using Peptidosomes. A, B and C illustrate the experimental set-up whereas D, E and F display the results of the well scan. There is only an increase of luminescence (≥ 40 RLU, pink), restricted to the Peptidosomes, if the encapsulated ReSt is cultivated within the SeSt (A, D). Cultures containing either SeSt (B, E) or encapsulated ReSt (C, F) served as negative controls.