Team:TU Dresden/Demonstrate

Mission Accomplished

Peptidosomes

Short Description

Peptidosomes are the new fundamental approach for generating and applying encapsulated bacteria. We are creating cages containing a liquid environment inside. The mesh-like structure of the cage allows the selective exchange of compounds via diffusion. Therefore, we are able to benefit from the entrapped cells’ abilities, while still ensuring that they are not released into their surroundings. Peptidosomes can be further enhanced by incorporating magnetic or biological beads – which are also functionalized with proteins – into their peptide-based fibrillary shell.

Achievements

We proved that we are able to produce stable Peptidosomes with the sizes of 1 µL to 20 µL (Figure 1). Furthermore we showed that diffusion between the inside of the Peptidosomes and the environment is possible (Figure 2). This was crucial because it is necessary to make fresh nutrients available for the organism and to allow the release of secreted molecules of interest out of the peptidosome while keeping the bacteria inside.

As demonstrated with different methods, we are able to encapsulate bacteria inside the Peptidosome and detect them, i.e. with the plate reader well-scan technique (Figure 3). In addition we proved that the bacteria can also grow inside the Peptidosome.

By trapping Dynabeads inside the shell and attaching molecules to them we managed to decorate the surface (Figure 4).

Different sizes of Peptidosomes
Figure 1: Different sizes of Peptidosomes
Diffusion between the inside of the Peptidosomes and the surrounding environment.
Figure 2: Diffusion between the inside of the Peptidosomes and the surrounding environment. The peptidosomes are shown directly after transfer in water (transparent liquid) or LB medium (yellowish liquid) and after 30 minutes.
Well scan of Peptidosome
Figure 3: Well scan of a Peptidosome with loaded bacteria expressing sfGFP: A results of the well-scan measurement for the detection of fluorescence is shown. If no signal is detected, the field of the matrix is green, otherwise red.
Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (WB) or LB media (LB)
Figure 4: Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (WB) or LB media (LB)

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 a 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. Besides the detection range, we analyzed the sensitivity of the biosensor for these specific compounds in several dose-response experiments shown in Figure 5. Furthermore, we demonstrated the applicability of the biosensor when encapsulated into Peptidosomes. As depicted in Figure 6, 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.

Figure 5: Dose-Response Curves of the Biosensor
Figure 5: 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 6: Encapsulation of the biosensor into Peptidosomes
Figure 6: 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 Parts Registry) enables an organism-independent, straightforward approach to identifying the best combination of SP with any protein of interest.

Achievements

We evaluated and proved the applicability of a powerful toolbox to quickly screen via a high throughput procedure for improved secretion of proteins in bacteria - the Signal Peptide Toolbox. It combines combinatorial construction with highly reproducible, quantitative measurements to maximize secretion levels.

We applied the Signal Peptide Toolbox to three different proteins. Via demonstrating the secretion of sfGFP, amyE and mCherry, and identifying the most potent SP-protein combinations for each of them (Figure 7, 8, 9), we proved the applicability of this powerful toolbox.

Figure 9: Sequenced signal peptides in front of mCherry. Fold change in secretion efficiency (fluorescence) over wild type. Depicted candidates were identified by sequencing.
Figure 8: Sequenced signal peptides in front of amyE. Fold change in secretion efficiency (amylase activity) over wild type. Depicted candidates were identified by sequencing.
Figure 7: Sequenced signal peptides in front of sfGFP. Fold change in secretion efficiency (fluorescence) over wild type. Depicted candidates were identified by sequencing.

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

Figure 11: Three color stages of the EV. All agar plates shown contain X-Gal and IPTG. A The EV without any inserts. B The EV with an inserted SP and gene of interest. C The EV with RPFsyn2 and an inserted gene of interest.
An example picture to show how to include them.
Figure 10: Vector map of the EV. The MCS is indicated in colors, grey elements refer to features necessary for cloning in E. coli and the white elements refer to B. subtilis specific vector parts.

We built a unique multiple cloning site which allows for easy insertion of both, a promoter and two basic or composite parts - the Evaluation Vector. The distinct features of the Evaluation Vector provide an easy cloning and screening workflow (Figure 10).

Additionally, the insertion of reporters to identify positive replacements by insert integration allow for a quick cloning and screening procedure in Escherichia coli. All three different stages of the insertion of expression units can be identified via a three-color scheme easily (Figure 11).

Furthermore, we proved the applicability and functionality of the Evaluation Vector as we evaluated it in the course of the Signal Peptide Toolbox.

Secretion

Short Description

In combining 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 setup 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 12 we assayed the fluorescence of the supernatant containing mCherry SpyCatcher/SpyTag constructs, which surpasses the wild type by far. The typical red color of mCherry is even visible in the supernatant under day light conditions (Figure 13).

We demonstrated the functionality of our SpyTag/SpyCatcher system via SDS-PAGE (Figure 14). Upon 4 hr 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 12: Endpoint measurement of the fluorescence from supernatants.
Figure 12: 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 13: Supernatants of <i>B. subtilis</i> cultures.
Figure 13: 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 14: SDS gel with crude and purified supernatants.
Figure 14: 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 μl 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 hr, 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 signaling molecules. This part of EncaBcillus is focused on proving 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 15). 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 16) we applied it to Peptidosomes (Figure 17). 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 15: Growth of the ReSt (A) and ComX-dependent promoter activity of P<sub><i>srfA</i></sub> (B) in transformation medium (MNGE).
Figure 15: 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 16: Co-culture of sender strain (SeSt) and receiver strain (ReSt) using ThinCert™ cell culture inserts.
Figure 16: 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 17: Co-culture of sender strain (SeSt) and receiver strain (ReSt) using Peptidosomes.
Figure 17: 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.