Revision as of 14:06, 1 November 2017

It´s a trap!

Short Description

Peptidosomes are the new fundamental approach for generating and applying encapsulated bacteria. By the creation of spherical compartments containing a liquid environment inside, bacteria are still able to grow and fulfil a given task. The mesh-like structure of the sphere allows the selective exchange of compounds via diffusion, but holds the bacteria trapped inside. 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 themselves can be functionalized with proteins – into their peptide-based fibrillary shell.

Background

Bacteria are omnipresent in biotechnology and applied projects. They can be used as hosts to produce nearly any biological compound of interest such as: drugs, vaccines, enzymes, antibiotics or even fuels and solvents. Their fast life cycle and comparable low requirements of living conditions highlight their industrial relevance. Over the last decades, the main focus to increase yields laid on extensive metabolic engineering and optimizing growth conditions.

Yet, there are more aspect which need to be considered when producing a compound of interest. First, where is the product of interest found: inside of the producing strain or will it be secreted to the surrounding media? Second, what is necessary to separate the valuable end-product from the bacteria? And maybe most important, how to assure a safe use of genetically engineered production strains? If you would like to know more on encapsulated bacteria in Peptdisomes and boosting the production of a compound of interest check out our: Signal Peptide Toolbox and Peptide Secretion sections.

To address these major biological and technical questions, the TU Dresden iGEM team presents EncaBcillus. Using Bacillus subtilis as model organism we introduce a new fundamental approach for cultivation of bacteria: the Peptidsomes. These Peptidosmes are buildup of self-assembled Fmoc dipeptide phenylalanines (Fmoc-FF) and are able to form spherical cages. The cages hold back the bacteria from the surrounding but are freely diffusible for smaller molecules. We show, that B. subtilis is able to grow, while encapsulated inside of the Pepdiosomes and demonstrate the applicability of two (fluorescence and luminescence) reports for the use with Pepdiosomes.

While evaluating this new method of bacterial immobilization, we established two applications using Peptidsomes:

Design

Fmoc-FF

Peptidosome creation

Exposing Fmoc-FF to gaseous CO₂ was the method of choice we wanted to exploit for the creation of the Peptidosomes. The challenge was to build a closed round cage formed by an Fmoc-FF membrane surrounding a liquid core. For this, we used an ultra-hydrophobic surface. When deposited on an ultra-hydrophobic surface, a droplet of water will minimize the contact with this surface. This will result in a droplet with a contact angle higher than 160°. In other words, the droplet will remain stable almost forming a perfect sphere [8].

To prepare a Peptidosome, a droplet of Fmoc-FF solution is placed on the mentioned membrane, keeping its spherical shape due to the ultrahydrophobicity. Afterwards the droplet is exposed directly with CO₂. The ultrahydrophobic PTFE membrane allows CO₂ to reach the entire surface of the droplet, even the small contact surface on the permeable membrane.

Exposing with CO₂ causes a pH change in the droplet. Carbonic acid is formed when the gas comes into contact with water. The carbonic acid subsequently dissociates into bicarbonate ions and hydrogen ions (Figure 2).

Cresol red was added as pH indicator to follow the drop of the pH during the experiment. A red / violet coloration can be seen in the basic pH. With acidification to a pH below 7.0, the solution turns yellow.

The Fmoc-FF solution has a pH of 10.5. Exposing with CO₂ reduces the pH value. The carboxy group of the Fmoc-FF is protonated, neutralizing the negative charge and triggering the self-assembling of the dipeptide molecules. Since the CO₂ only comes into contact with the surface of the droplet, a fibrillar network is formed only there, while the inside of the droplet retains its liquid state (Figure 3).

Reaction in Peptidosomes — **Figure 2: Chemical reaction in Peptidosome upon CO₂ exposure** The reaction of carbon dioxide and water leads to the formation of carbonic acid, which dissociates into bicarbonate ions and hydrogen ions. All reactions are reversible

Schematic representation of the change of the Fmoc-FF molecule during Peptidosome production — **Figure 2: Chemical reaction in Peptidosome upon CO₂ exposure** The reaction of carbon dioxide and water leads to the formation of carbonic acid, which dissociates into bicarbonate ions and hydrogen ions. All reactions are reversible

Bacteria encapsulated in Peptidosomes

Results

Characterization of Peptidosomes

Before we could encapsulate bacteria, we had to establish a robust and reproducible protocol of Peptidosome generation under lab conditions. To tackle this task, we used pH indicator cresol red solutions to visualise generated Peptidosomes (Figure 5). In addition to ease the handling of the Peptidosomes, the pH indicator colour also correlated with the status of the Peptidosomes formation. Prior the CO₂ exposure the indicator was red (Figure 5, A), after sufficient CO₂ exposure (will be discussed later) the solution changed to yellow (Figure 5, B), indicating a self-assembled Fmoc-FF layer and thus ready-to-use Peptidosomes.

These generated Peptidosomes stayed stable after CO₂ treatment and we were able to transfer them into liquid filled petri-dishes or well plates.

After we having established the protocol for the Peptidosome creation, we evaluated if we could vary the size of the Peptidosomes. For testing this, we simply generated Peptidosomes of different volumes and could demonstrate successful created and stable Peptidosomes ranging from 1 to 20 µl (Figure 6).

For discovering the optimal exposure time to CO₂ we varied it between 30 sec and 20 min. We found out, that the ones with an exposure time of 10 min were the most stable ones, showing stability after several days, even under 37°C incubation with shaking (Figure 7).

**Figure 5: The color change of the Peptidosomes with pH indicator during production.** A Peptidosomes before exposure to CO₂ the pH indicator is red as a control, Peptidosomes without indicator are shown, these are transparent B Peptidosomes after the exposure, only the Peptidosomes with pH indicator changed color from red to yellow

Different sizes of Peptidosomes — **Figure 5: The color change of the Peptidosomes with pH indicator during production.** A Peptidosomes before exposure to CO₂ the pH indicator is red as a control, Peptidosomes without indicator are shown, these are transparent B Peptidosomes after the exposure, only the Peptidosomes with pH indicator changed color from red to yellow

The final aim of the project is the encapsulation and cultivation of bacteria inside the Peptidosomes. For this, it is crucial that while the Fmoc-FF network should hold back the bacteria on the other side it should allow the interchange and communication with the surrounding environment. Thus, allowing fresh nutrients for the organism to enter the inside, and/or to allow the release of secreted molecules to the outside of the Peptidosomes.

**Figure 8: 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.

Proving that diffusion between the inside of the Peptidosome and the surrounding is possible, we next tested if diffusion between two Peptidosomes which are in direct contact is possible. For this experiment only one of the two connected Peptidosomes contained the coloured pH indicator solution. Over the lapse of 1.5 hours, it was possible to observe a diffusion of the colour between both peptidosome until it reached an equilibrium (Figure 9). This proved that there is intercommunication, between the two Peptidosomes.

**Figure 9: Diffusion between Peptidosomes** Different time points of the experiment are shown. The yellow peptidosome (with pH indicator) was fused with an empty Peptidosome (transparent). The color change was then observed over time (1.5 h).

Additionally, to the previous described method of Peptidosome generation, we also tried a microinjection-technique. By doing so, we injected a coloured solution to the inside of the Peptidosome (Figure 10). To close the membrane hole that was introduced by the thin glass capillary, the Peptidosome was after injection again exposed to CO₂ for 5 min. Followed by this procedure, the Peptidosome was transferred to water and as shown in Figure 10 E) the peptidosome was stable.

**Figure 10: Microinjection of stained water into a Peptidosome** A), B), C), D): injection of the solution with a glass capillary E) in water tranferred Peptidosome of this method.

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 Fmoc-FF. One aspect tested was, whether the bacteria can use Fmoc-FF as a nitrogen source. And also, if the organism can survive the process of encapsulation, since the bacterial pellet is resuspended in the (high) alkaline Fmoc-FF solution.

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

To test, if B. subtilis can degrade Fmoc-FF, we tested bacterial growth on Jensen’s medium agar plates. This medium has all the basic nutrients that bacteria needs for growth, with exception of nitrogen.

The experimental used agar plates were set-up was following:

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

Treatment 1 was a negative control, since B. subtilis (wildtype) cannot grow 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

Comparison of B.subtilis cell growth at different pH values — **Figure 11: Comparison of **B.subtilis** cell growth at different pH values** Shown is the change in optical density over the cultivation period. Each graph represents a culture approach. In all batches, NaOH or water (red dot) was induced after one hour to adjust a certain pH value.

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.

You can find the detailed protocols here.

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. Please check ot the concerning protocol.

In figure 12 examples of the well scans of different samples are displayed.

Overview Well-scans of the Plate Reader Assay — **Figure 12: Overview of well-scans of the Plate Reader Assay** The results of the well-scan measurements for the detection of fluorescence and luminescence are shown. If no signal is detected, the field of the matrix is green, otherwise red.

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

**Figure 13: Peptidosome with encapsulated fluorescent 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.

Table 1: Results growth experiment

Time [h]	0	3.5	7
Colonies	370.67 ± 57.42	593.33 ± 74.44	Lawn

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.

Cryo-SEM

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.

**Figure 14: SEM pictures** A) whole Peptidosome B),C) Details of the membrane D),E),F) Details of the bacteria trapped in the Peptidosome membrane

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.

Table 2: Results growth experiment with different treatments

Treatment	Normal	Washing	pH treatment
Colonies	203.7 ± 309.1	0.7 ± 1.2	1.7 ± 1.9

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.

**Figure 15: SEM pictures** A), B) Membrane overview C),D) Membrane details; A), C) washings steps B), D) pH treatment

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 15 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 16).

**Figure 16: Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer.** A) transmitted light B) fluorenscence

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 17).

**Figure 17: Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (A) or LB media (B)**

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.

References

[1]	K. Tao, A. Levin, L. Adler-Abramovich, and E. Gazit, “Fmoc-modified amino acids and short peptides: simple bio-inspired building blocks for the fabrication of functional materials.,” Chem. Soc. Rev., vol. 45, pp. 3935–3953, 2016.
[2]	M. Reches and E. Gazit, “Casting Metal Nanowires Within Discrete Self-Assembled Peptide Nanotubes,” Science (80-.), vol. 300, no. 5619, pp. 625–627, 2003.
[3]	C. H. Görbitz, “The structure of nanotubes formed by diphenylalanine, the core recognition motif of Alzheimer’s β-amyloid polypeptide,” Chem. Commun., no. 22, pp. 2332–2334, 2006.
[4]	M. Reches and E. Gazit, “Self-Assembly of Peptide Nanotubes and Amyloid-like Structures by Charged-Termini-Capped Diphenylalanine Peptide Analogues,” Isr. J. Chem., vol. 45, pp. 363–371, 2005.
[5]	V. Jayawarna et al., “Nanostructured hydrogels for three-dimensional cell culture through self-assembly of fluorenylmethoxycarbonyl-dipeptides,” Adv. Mater., vol. 18, no. 5, pp. 611–614, 2006.
[6]	C. Tang, A. M. Smith, R. F. Collins, R. V. Ulijn, and A. Saiani, “Fmoc-Diphenylalanine Self-Assembly Mechanism Induces Apparent pK a Shifts,” Langmuir, vol. 25, no. 16, pp. 9447–9453, 2009.
[7]	H. G. Braun and A. Z. Cardoso, “Self-assembly of Fmoc-diphenylalanine inside liquid marbles,” Colloids Surfaces B: Biointerfaces, vol. 97, pp. 43–50, 2012.
[8]	M. Zhang, S. Feng, L. Wang, and Y. Zheng, “Lotus effect in wetting and self-cleaning,” Biotribology, vol. 5, pp. 31–43, Mar. 2016.

@@ Line 101: / Line 101: @@
-<div <div class="contentbox">
+<div <div <div class="contentbox"><div <div class="contentbox">
 <h1 class="box-heading">Results</h1>
 <h3>Characterization of Peptidosomes </h3>
-<p>Before we could encapsulate bacteria, we had to establish a robust and reproducible protocol of Peptidosome generation under lab conditions. To tackle this task, we used pH indicator cresol red solutions to visualise generated Peptidosomes (Figure 4). In addition to ease the handling of the Peptidosomes, the pH indicator colour also correlated with the status of the Peptidosomes formation. Prior the CO<sub>2</sub> exposure the indicator was red (Figure 4, A), after sufficient CO<sub>2</sub> exposure (will be discussed later) the solution changed to yellow (Figure 4, B), indicating a self-assembled Fmoc-FF layer and thus ready-to-use Peptidosomes.</p>
+<p>Before we could encapsulate bacteria, we had to establish a robust and reproducible protocol of Peptidosome generation under lab conditions. To tackle this task, we used pH indicator cresol red solutions to visualise generated Peptidosomes (Figure 5). In addition to ease the handling of the Peptidosomes, the pH indicator colour also correlated with the status of the Peptidosomes formation. Prior the CO<sub>2</sub> exposure the indicator was red (Figure 5, A), after sufficient CO<sub>2</sub> exposure (will be discussed later) the solution changed to yellow (Figure 5, B), indicating a self-assembled Fmoc-FF layer and thus ready-to-use Peptidosomes.</p>
 <p>These generated Peptidosomes stayed stable after CO<sub>2</sub> treatment and we were able to transfer them into liquid filled petri-dishes or well plates.</p>
-<p>After we having established the protocol for the Peptidosome creation, we evaluated if we could vary the size of the Peptidosomes. For testing this, we simply generated Peptidosomes of different volumes and could demonstrate successful created and stable Peptidosomes ranging from 1 to 20 µl (Figure 5).</p>
+<p>After we having established the protocol for the Peptidosome creation, we evaluated if we could vary the size of the Peptidosomes. For testing this, we simply generated Peptidosomes of different volumes and could demonstrate successful created and stable Peptidosomes ranging from 1 to 20 µl (Figure 6).</p>
 <p>
-For discovering the optimal exposure time to CO<sub>2</sub> we varied it between 30 sec and 20 min. We found out, that the ones with an exposure time of 10 min were the most stable ones, showing stability after several days, even under 37°C incubation with shaking (Figure 6). </p>
+For discovering the optimal exposure time to CO<sub>2</sub> we varied it between 30 sec and 20 min. We found out, that the ones with an exposure time of 10 min were the most stable ones, showing stability after several days, even under 37°C incubation with shaking (Figure 7). </p>
 <figure>
 <figure class="makeresponsive floatleft" style="width:35.5%;">
@@ Line 117: / Line 117: @@
             alt="The color change of the Peptidosomes with pH indicator during production"
             class="zoom">
-       <figcaption><b>Figure 4: The color change of the Peptidosomes with pH indicator during production.</b> <b>A</b> Peptidosomes before exposure to CO<sub>2</sub> the pH indicator is red as a control, Peptidosomes without indicator are shown, these are transparent <b>B</b> Peptidosomes after the exposure, only the Peptidosomes with pH indicator changed color from red to yellow</figcaption>
+       <figcaption><b>Figure 5: The color change of the Peptidosomes with pH indicator during production.</b> <b>A</b> Peptidosomes before exposure to CO<sub>2</sub> the pH indicator is red as a control, Peptidosomes without indicator are shown, these are transparent <b>B</b> Peptidosomes after the exposure, only the Peptidosomes with pH indicator changed color from red to yellow</figcaption>
 </figure>
 <figure style="width:29%;" class="makeresponsive floatleft">
@@ Line 123: / Line 123: @@
             alt="Different sizes of Peptidosomes"
             class="zoom">
-       <figcaption><b>Figure 5: Different sizes of Peptidosomes</b> </figcaption>
+       <figcaption><b>Figure 6: Different sizes of Peptidosomes</b> </figcaption>
 </figure>
 <figure class="makeresponsive floatleft" style="width:35.5%;">
@@ Line 129: / Line 129: @@
             alt="Peptidosomes generated with different exposure times."
             class="zoom">
-       <figcaption><b>Figure 6: Peptidosomes generated with different exposure times. </b> Peptidosomes are shown, which were exposed to CO<sub>2</sub> for 30 sec, 3 min, 5 min, 10 min or 20 min.</figcaption>
+       <figcaption><b>Figure 7: Peptidosomes generated with different exposure times. </b> Peptidosomes are shown, which were exposed to CO<sub>2</sub> for 30 sec, 3 min, 5 min, 10 min or 20 min.</figcaption>
      </figure>
@@ Line 139: / Line 139: @@
             alt="Diffusion between the inside of the Peptidosomes and the surrounding environment."
             class="zoom">
-       <figcaption><b>Figure 7: Diffusion between the inside of the Peptidosomes and the surrounding environment. </b> The Peptidosomes are shown directly after transfer in water (transparent liquid) or LB medium (yellowish liquid) and after 30 minutes.</figcaption>
+       <figcaption><b>Figure 8: Diffusion between the inside of the Peptidosomes and the surrounding environment. </b> The Peptidosomes are shown directly after transfer in water (transparent liquid) or LB medium (yellowish liquid) and after 30 minutes.</figcaption>
 </figure>
 <p></p>
 <p>Using a straight forward experiment, applying 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 medium and monitored the time the pH indicator needed to diffuse outside of the Peptidosomes. </p>
-<p>The yellow color of both Paptidosomes (in water and LB) faded over time (Figure 7). In water (left panel), the yellow color completely vanished after about 25-30 minutes. In LB the same effect was visible after 45-50 minutes (right panel).
+<p>The yellow color of both Paptidosomes (in water and LB) faded over time (Figure 8). In water (left panel), the yellow color completely vanished after about 25-30 minutes. In LB the same effect was visible after 45-50 minutes (right panel).
 These observations met our expectations, as the Fmoc-FF membrane is a network of tiny pores, rather than a closed shell. These pores allow the exchange with the surrounding liquid.</p>
 </figure>
-<p>Proving that diffusion between the inside of the Peptidosome and the surrounding is possible, we next tested if diffusion between two Peptidosomes which are in direct contact is possible. For this experiment only one of the two connected Peptidosomes contained the coloured pH indicator solution. Over the lapse of 1.5 hours, it was possible to observe a diffusion of the colour between both peptidosome until it reached an equilibrium (Figure 8). This proved that there is intercommunication, between the two Peptidosomes. </p>
+<p>Proving that diffusion between the inside of the Peptidosome and the surrounding is possible, we next tested if diffusion between two Peptidosomes which are in direct contact is possible. For this experiment only one of the two connected Peptidosomes contained the coloured pH indicator solution. Over the lapse of 1.5 hours, it was possible to observe a diffusion of the colour between both peptidosome until it reached an equilibrium (Figure 9). This proved that there is intercommunication, between the two Peptidosomes. </p>
   <p> </p>
   <figure class="makeresponsive" style="width: 65%; display: flex; align-items: center; justify-content: center; flex-direction: column;"">
@@ Line 153: / Line 153: @@
             alt="Diffusion between Peptidosomes"
             class="zoom">
-       <figcaption><b>Figure 8: Diffusion between Peptidosomes </b> Different time points of the experiment are shown. The yellow peptidosome (with pH indicator) was fused with an empty Peptidosome (transparent). The color change was then observed over time (1.5 h).</figcaption>
+       <figcaption><b>Figure 9: Diffusion between Peptidosomes </b> Different time points of the experiment are shown. The yellow peptidosome (with pH indicator) was fused with an empty Peptidosome (transparent). The color change was then observed over time (1.5 h).</figcaption>
      </figure>
 <p> </p>
-<p>Additionally, to the previous described method of Peptidosome generation, we also tried a microinjection-technique. By doing so, we injected a coloured solution to the inside of the Peptidosome (Figure 9). To close the membrane hole that was introduced by the thin glass capillary, the Peptidosome was after injection again exposed to CO<sub>2</sub> for 5 min. Followed by this procedure, the Peptidosome was transferred to water and as shown in Figure 9 E) the peptidosome was stable. </p>
+<p>Additionally, to the previous described method of Peptidosome generation, we also tried a microinjection-technique. By doing so, we injected a coloured solution to the inside of the Peptidosome (Figure 10). To close the membrane hole that was introduced by the thin glass capillary, the Peptidosome was after injection again exposed to CO<sub>2</sub> for 5 min. Followed by this procedure, the Peptidosome was transferred to water and as shown in Figure 10 E) the peptidosome was stable. </p>
 <figure class="makeresponsive" style="width: 60%; display: flex; align-items: center; justify-content: center; flex-direction: column;">
        <img src="https://static.igem.org/mediawiki/2017/b/be/T--TU_Dresden--microinjection.png"
             alt="Microinjection of stained water into a Peptidosome"
             class="zoom">
-       <figcaption><b>Figure 9: Microinjection of stained water into a Peptidosome </b> A), B), C), D): injection of the solution with  a glass capillary E) in water tranferred Peptidosome of this method.</figcaption>
+       <figcaption><b>Figure 10: Microinjection of stained water into a Peptidosome </b> A), B), C), D): injection of the solution with  a glass capillary E) in water tranferred Peptidosome of this method.</figcaption>
      </figure>
 <hr>
@@ Line 190: / Line 190: @@
             alt="Comparison of B.subtilis cell growth at different pH values"
             class="zoom">
-       <figcaption><b>Figure 10: Comparison of <i><b>B.subtilis</b></i> cell growth at different pH values </b> Shown is the change in optical density over the cultivation period. Each graph represents a culture approach. In all batches, NaOH or water (red dot) was induced after one hour to adjust a certain pH value.</b> </figcaption>
+       <figcaption><b>Figure 11: Comparison of <i><b>B.subtilis</b></i> cell growth at different pH values </b> Shown is the change in optical density over the cultivation period. Each graph represents a culture approach. In all batches, NaOH or water (red dot) was induced after one hour to adjust a certain pH value.</b> </figcaption>
      </figure>
@@ Line 209: / Line 209: @@
 <p>For the first experiments of encapsulation, we used the <i>B. subtilis</i> strains TMB4131 W168 <i>lacA::erm Pveg_sfGFP</i> and TMB3090 W168 <i>sacA::cat Pveg_luxABCDE</i>. 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. Please check ot the concerning <a   href="https://2017.igem.org/Team:TU_Dresden/Experiments">protocol</a>.</p>
-<p>In figure 11 examples of the well scans of different samples are displayed.</p>
+<p>In figure 12 examples of the well scans of different samples are displayed.</p>
 <p></p>
 <figure class="makeresponsive" style="width: 60%; display: flex; align-items: center; justify-content: center; flex-direction: column;">
@@ Line 215: / Line 215: @@
             alt="Overview Well-scans of the Plate Reader Assay "
             class="zoom">
-       <figcaption><br><b>Figure 11: Overview of well-scans of the Plate Reader Assay </b> The results of the well-scan measurements for the detection of fluorescence and luminescence are shown. If no signal is detected, the field of the matrix is green, otherwise red.</figcaption>
+       <figcaption><br><b>Figure 12: Overview of well-scans of the Plate Reader Assay </b> The results of the well-scan measurements for the detection of fluorescence and luminescence are shown. If no signal is detected, the field of the matrix is green, otherwise red.</figcaption>
      </figure>
      <br>
@@ Line 229: / Line 229: @@
             alt="Peptidosome with encapsulated fluorescent bacteria"
             class="zoom">
-       <figcaption><b>Figure 12: Peptidosome with encapsulated fluorescent bacteria </b></figcaption>
+       <figcaption><b>Figure 13: Peptidosome with encapsulated fluorescent bacteria </b></figcaption>
 </figure>
-<p>The encapsulation of <i>B. subtilis</i> expressing sfGFP in a constitutive way was also demonstrated by the fluorescence microscopy. In this experiment, the bacteria were encapsulated as described. As shown in Figure 12, the Peptidosome emitted green light what proved the existence of sfGFP-expressing bacteria inside of the Peptidosome. </p>
+<p>The encapsulation of <i>B. subtilis</i> expressing sfGFP in a constitutive way was also demonstrated by the fluorescence microscopy. In this experiment, the bacteria were encapsulated as described. As shown in Figure 13, the Peptidosome emitted green light what proved the existence of sfGFP-expressing bacteria inside of the Peptidosome. </p>
 </figure>
@@ Line 293: / Line 293: @@
             class="makeresponsive zoom">
      </figure>
-     <figcaption> <b>Figure 13: SEM pictures</b> A) whole Peptidosome B),C) Details of the membrane D),E),F) Details of the bacteria trapped in the Peptidosome membrane </figcaption>
+     <figcaption> <b>Figure 14: SEM pictures</b> A) whole Peptidosome B),C) Details of the membrane D),E),F) Details of the bacteria trapped in the Peptidosome membrane </figcaption>
     </figure>
@@ Line 345: / Line 345: @@
      </figure>
-     <figcaption> <b>Figure 14: SEM pictures</b> A), B) Membrane overview C),D) Membrane details; A), C) washings steps B), D) pH treatment </figcaption>
+     <figcaption> <b>Figure 15: SEM pictures</b> A), B) Membrane overview C),D) Membrane details; A), C) washings steps B), D) pH treatment </figcaption>
     </figure>
 <p></p>
-<p>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)) </p>
+<p>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 15 A), B)) </p>
 <p>Only on the Peptidosome with the low pH treatment, a cell that was not integrated in the membrane was observed (D). </p>
 <p>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. </p>
@@ Line 364: / Line 364: @@
    </video>
-<p>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 15). </p>
+<p>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 16). </p>
 <br>
 <figure class="makeresponsive" style="width: 65%; display: flex; align-items: center; justify-content: center; flex-direction: column;">
@@ Line 370: / Line 370: @@
             alt="Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer."
             class="zoom">
-       <figcaption><b>Figure 15: Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer. </b> A) transmitted light B) fluorenscence </figcaption>
+       <figcaption><b>Figure 16: Dynabeads labeled with histidine-tagged GFP in the Binding/Wash buffer. </b> A) transmitted light B) fluorenscence </figcaption>
      </figure>
 <br>
-<p>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 16). </p>
+<p>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 17). </p>
 <br>
 <figure class="makeresponsive" style="width: 50%; display: flex; align-items: center; justify-content: center; flex-direction: column;">
@@ Line 379: / Line 379: @@
             alt="Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (A) or LB media (B)"
             class="zoom">
-       <figcaption><b>Figure 16: Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (A) or LB media (B) </b> </figcaption>
+       <figcaption><b>Figure 17: Peptidosome with Dynabeads labeled with His-Tag GFP in Binding/Wash Buffer (A) or LB media (B) </b> </figcaption>
      </figure>
 <p>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. </p>

Difference between revisions of "Team:TU Dresden/Project/Peptidosomes"