With a really straight forward experiment, including the help of the pH-indicator we were able to prove that a diffusion between the inside of the peptidosome and the surrounding environment is possible. We transferred Peptidosomes to water or LB-Media and measured the time it took for the colour to disappear, or in other words, the time it takes for the coloured solution inside the cage to diffuse out of it.

Proving that diffusion between the inside of the peptidosome and the environment is possible, we wanted to know next if diffusion between two peptidosomes which are in direct contact is possible. For this experiment only one of the two connected peptidosomes contained coloured solution. Over the lapse of around 1.5 hours, it was possible to observe how the colour was disseminating to the non-coloured peptidosome until it reached an equilibrium (Figure X), proving that there is diffusion, or intercommunication, between the two peptidosomes.

Additionally, to the previous described method, we used a microinjection-technique to inject a coloured solution to the inside of the peptidosome (figure XXX). To close the hole that was probably opened on the membrane by the introducing the glass capillary, the peptidosome was exposed again for 5 min to CO₂. Afterwards it was transferred to water. As shown in figure XXX D) the peptidosome was stable.

2. Encapsulation of Bacillus subtilis in Peptidosomes

Before the encapsulation of B. subtilis inside the peptidosomes, it was necessary to perform experiments to study possible interactions between the organism and the dipeptide, for example, whether the bacteria can use Fmoc-FF as a nitrogen source, or if the organism can survive the process of encapsulation, since the bacterial pellet is resuspended in the alkaline Fmoc-FF solution.

Can Fmoc-FF use B. subtilis as a nitrogen source?

Special culture plates were created using Jensen’s medium. This type of medium has all the basic nutrients that bacteria needs for its development, with exception of nitrogen.

Four treatments were tested:

• 1) Nitrogen free Jensen’s medium
• 2) Jensen’s medium supplemented with nitrogen (adding Fe [III] ammonium citrate and Potassium glutamate)
• 3) Jensen’s medium mixed with Fmoc-FF
• 4) Jensen’s medium with Fmoc-FF added over the dried plates

Treatment 1 was a negative control, since the strain of B. subtilis (wildtype) cannot develop in absence of nitrogen, so no growth at all was expected. Treatment 2 was the positive control: with the nitrogen supplement, B. subtilis should be able to grow. These expectations corresponded with reality: colonies grew in treatment 2 but none in treatment 1. Treatments 3 and 4 did not result in colony growth either, meaning that the Fmoc-FF cannot be used as a source of nitrogen for the bacteria.

Tolerance of B. subtilis against alkaline pH

The experiment to test the tolerance was performed as follows: B. subtilis culture was divided in four treatments, varying the pH of the culture in each one; 7 (normal growth condition as a control), 8, 9, and 10. NaOH was used to induce the different changes in the pH for each treatment, and was added after one hour of cultivation. The experiment was carried out in a plate reader to follow the change of the optical density (OD₆₀₀) which correlates with bacterial growth.

The graph shows the growth of the organisms under each condition. It can be observed that from pH 7 to 9, no significant change in the growth is observed. However, under pH 10, the organism stops its growth. To check if the bacteria was still viable after the high pH treatment, it was transferred to growth plates with neutral pH. There, it was observed that B. subtilis was able to recover and growth normally, meaning that the organism can survive the process of encapsulation.

Bacillus subtilis in Peptidosomes

Once it was proven that B. subtilis can survive the designed encapsulation process, and that the organism cannot use the building block Fmoc-FF as a nitrogen source, experiments to entrap the bacteria started.

For this we resuspended an appropriate amount of bacteria in the Fmoc-FF solution. Afterwards droplets of this were deposited on the ultrahydrophobic membrane and exposed to CO₂ for 10 minutes and afterwards transferred to water or LB media.

Well Scan experiment

For the first experiments of encapsulation, we used the B. subtilis strains TMB4131 W168 lacA::erm Pveg_sfGFP and TMB3090 W168 sacA::cat Pveg_luxABCDE. The first strain has the characteristic of expressing sfGFP in a constitutive way, which was useful to prove the presence of bacteria in the peptidosomes by detecting the fluorescence expressed by the cells. TMB3090 expresses luciferase in a constitutive way, which makes a detection of a luminescence signal possible. We performed a plate reader assay using the well-scan-mode. In this mode, the whole well is scanned to detect the exact position of a signal, either fluorescence or luminescence. Its absence is displayed in a map with a green colour, while the position of the fluorescence/luminescence source appears as red.

In figure X examples of the well scans of different samples are displayed.

The well scan maps appear completely green where fluorescence/luminescence is absent, i.e. water and empty peptidosome. Wells containing a sample of the day culture and lyophilized eGFP solved in water show a red colour in the whole map. However, a localized red spot over a green background is observed where the source of fluorescence/luminescence is contained: the cells trapped inside the peptidosomes. This shows that when the peptidosome with cells inside is transferred to water, bacteria cannot diffuse away, but its kept contained within the structure, resulting in a localized red signal.

Fluorescence microscopy

The encapsulation of B. subtilis expressing sfGFP in a constitutive way was also demonstrated by the fluorescence microscopy. In this experiment, the bacteria were encapsulated as described. As seen in Figure X, the peptidosome emitted green light what proved the existence of sfGFP-expressing bacteria.

The Growth of B.subtilis in Peptidosomes

The method we used to check the growth and reproduction of bacteria inside the peptidosome was performed by generating peptidosomes loaded with a known amount of bacteria. Some peptidosomes were plated on LB agar right after being generated, while other peptidosomes were incubated in LB broth at 37°C for 3.5 and 7 hours and afterwards plated and incubated overnight. An increase in the number of colonies formed by the incubated peptidosomes means that bacteria can grow inside the structure. The result is shown in the next table.

As observed on the results, the number of colonies in the plates are increasing after 3.5 h of incubation, until finally after 7 hours it is not possible to count the amount of colonies anymore, meaning that the cells can reproduce inside the cages, therefore, peptidosomes are indeed suitable for the establishment of bacterial cultures.

When performing that experiment, we discovered bacterial growth in the supernatant where the peptidosomes were incubated. To figure out the reason for this, cryo-Scanning Electron Microscopy (cryo-SEM) was used to image bacteria-loaded peptidosomes. The results are showed below.

As observed in the images, some cells are completely or only partially integrated into the membrane, composed by a mesh of fibers. This cells found on the outer surface of the membrane, if released, can be responsible for the bacterial growth on the supernatant. Therefore we designed a second growth experiment, in which also the whole supernatant of each peptidosome was plated with the purpose of observing the amount of bacteria released there.

Two extra treatments were tested, the first one consisted in adding “washing” steps, meaning that the peptidosomes were transferred twice to fresh media before their incubation. We hypothesized that the bacteria on the outer membrane would be released and left behind after the transfers. The second treatment that we tested consisted in pre-incubating the peptidosome in LB broth adjusted to have an acid pH, this with the finality of closing the membrane to entrap even more the cells, triggering the self-assembly of the unreacted Fmoc-FF solution still present in the peptidosome.

By introducing the treatments, the number of colonies was reduced from several hundreds to an average of .7 for the washing steps and 1.7 for the low pH treatment. After analysing the results, we concluded that the washing steps are important to reduce the number of cells released in the supernatant, whereas the treatment with acid broth does not add a significant effect on this. In that way, by introducing the washing steps and also reducing the concentration of cells in the peptidosomes, the amount of bacteria present on the outer surface of the membrane was importantly diminished, fact that was corroborated by performing a second cryo-SEM and showed below.

In the second measurement the number of detectable bacteria that lie or are integrated on the membrane is significantly lower. Only a few individual fibers are visible and the surface is wrinkled. (figure XX A), B))

Only on the peptidosome with the low pH treatment, a cell that was not integrated in the membrane was observed (D).

The results of the growth experiment with the additional treatments were confirmed microscopically. There was no significant improvement in the peptidosomes treated with acid LB medium compared to the sole integration of wash steps. Only a single cell, which could relieve from the peptidosome, was detected. This confirms the reduced number of colonies in the supernatant of the growth experiment when carrying out the additional treatment methods.

The one loosely attached cell might be a problem for some applications where the surrounding medium is expected to be free of bacteria, therefore, the manufacturing technique of peptidosomes should be adapted accordingly. An adequate method for this could be introducing the cells by microinjection, since there is not a chance that bacteria can be attached to the outer part of the membrane.

3. Surface decoration

The properties of self-assembled Fmoc-FF membrane allow tiny objects, such as Dynabeads, to be enveloped in the wall of the Peptidosome. Dynabeads envelopment enables the control of the Peptidosome movements in the magnetic field, that in combination with other features like producing strain encapsulation and small molecules or proteins diffusion, provides a powerful delivery tool, which can be used in various applications.

Nevertheless, the surface of DynaBeads can be decorated with His-Tag protein. For the “proof of principle” experiments His-Tag GFP were selected due to its availability and easy imaging procedure. First, the Invitrogen Protocol was optimized for our goals and tested for DynaBeads decoration with histidine-tagged GFP (figure X).

To prove the surface of the Dynabeads enveloped to the Peptidosome membrane availability for the decoration the labeling procedure was applied after the Peptidosome formation in both Binding/Wash Buffer and LB media (figure X).

In conclusion, here we proved the possibility of Peptidosome surface decoration with histidine-tagged molecules that can be used for various applications where enzymatic activity of the surface of Peptidosome or Peptidosome immobilization is required. Surface decoration protocol is compatible with almost any goals due to its flexibility, as the decoration procedure can be performed before and after Peptidosome formation in the specific Binding/Washing buffer or just in the LB media.