Abstract

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:

• An encapsulated β-lactam Biosensor
• Communication between encapsulated bacteria and their surrounding

Design

Fmoc-FF

For their many and very interesting properties, nanostructures are gaining great attention in the area of material sciences. Nanostructures are assemblies or clusters ordered within nanoscopic dimensions. Sometimes the building blocks of the nanostructures (with organic or inorganic sources) can assemble themselves, obeying several non-covalent forces that dictate their organization in supramolecular structures. These interactions can be for example hydrogen bonds, van der waals forces, aromatic interactions, among others [1]. We set our focus on the self-assembling building block Fmoc-FF (9-fluorenylmethoxycarbonyl diphenylalanine).

In 2003, while studying the mechanisms that direct the self-assembly of amyloid fibrils by short aromatic peptides, It was observed that the dipeptide diphenylalanine (FF), the core recognition motif of the Alzheimer’s β-amyloid polypeptide, was self-assembling into nanotubular structures in aqueous solution [2]. With the help of X-ray diffraction techniques, it was observed that self-assembly occurred due to the tight stacking of the phenyl rings in the dipeptide [3] Later, while studying the interactions in the process, the chemical group 9-fluorenylmethoxycarbonyl (Fmoc) was added at the N terminal of the dipeptide. The Fmoc cap was introducing an additional aromatic group, facilitating the self-assembly into typical amyloid-like fibrils [4].

Later an interesting property was discovered: it is possible to trigger the self-assembly of the dipeptide in solution. at a pH higher than 8, the dipeptide stayed in solution, but when slowly adding concentrated hydrochloric acid to reach pH levels below 8, a clear gel was formed [5].

At alkaline pH, the molecules are negatively charged and therefore repealing each other. If the pH drops, protonation of the molecules occurs, neutralizing the negative charge and permitting the self-assembly of the dipeptide into the aforementioned fibers [6]. In 2012 the formation of the mesh was induced by exposing a solution of Fmoc-FF with gaseous CO2 [7]. The gas reacts with the water and disassociates into bicarbonate ions and protons, which acidifies the solution and triggers the self-assembly of the dipeptide.

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Peptidosome creation

Using the mentioned method of Braun and Cardoso (2012) we build our Peptidosomes. The challenge was to build a closed round cage formed by an Fmoc-FF membrane surrounding a liquid core. For this we use 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 as 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.

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.

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.

Bacteria encapsulated in Peptidosomes

We wanted to characterize and prove the function of our Peptidosome with the use of Bacillus subtilis. This was a totally new approach since Fmoc-FF was never brought in contact with bacteria.

To encapsulate the bacteria we took samples of overnight cultures and day cultures prepared in LB media.

As soon as the day cultures had reached an OD600 between 0.2 and 0.6, the required amount of culture for the experiment was centrifuged for 5 min at 16,000 g. The supernatant was then discarded and the pellet resuspended in Fmoc-FF solution. Droplets of 15 μL of this solution were deposited on an ultrahydrophobic membrane and exposed with CO2.

For the growth experiments (LINK) Peptidosomes with an OD600 of 0.02 were created.

We performed multiple assays to prove the encapsulating and growth inside the Peptidsosome.

• Plate reader well scan
• Stereo fluorescence microscopie