Team:TU Dresden/Description

EncaBcillus – It’s a trap!

Synthetic biology wants to go beyond the pure biological by integrating concepts from chemistry or physics into the living world. At this interphase, our project wants to introduce Peptidosomes as a new fundamental approach for generating and applying encapsulated bacteria. These spheres possess advantageous properties like stability in different media and a mesh-like structure that allows for the selective exchange of compounds via diffusion. Therefore, we are able to benefit from the entrapped cells’ abilities, while ensuring that they are not released into their surroundings. Using the powerful genetics of Bacillus subtilis and its secretory capabilities we demonstrate communication and cooperation between separately encapsulated bacterial populations as well as the environment. Peptidosomes can be further enhanced by incorporating magnetic or biological beads – which can be functionalized with proteins – into their peptide-based shell. With this unique setup, we provide a whole new universe of applications to the iGEM community.

Get a quick overview about our project on our wiki's landing page or by checking out the poster we used to present our project during poster sessions and which was nominated for Best Poster!

Our Poster from the Giant Jamboree

Peptidosomes

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 fulfill 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.

Biosensor

Worldwide, multidrug-resistant germs 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, we developed a functional β-lactam biosensor in Bacillus subtilis. By the time these specified cells sense a compound of the β-lactam family, they will respond by producing a measurable luminescence signal. Here, we analyzed the detection range and sensitivity of the biosensor in response to six different β-lactam antibiotics. The evaluated Biosensor was then encapsulated into Peptidosomes to prove the concept of our project EncaBcillus. The trapping of engineered bacteria thus will allow for increased control and simplified handling, potentially raising the chances for their application e.g. sewage treatment plants.

Secretion

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.

Communication

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 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.

Signal Peptide Toolbox

Protein secretion is mainly orchestrated by Signal Peptides (SP). However, secretion efficiency is not determined by the SP but the combination of a SP with a specific protein. This necessitates establishing screening procedures to evaluate all possible combinations. We developed such an approach, the Signal Peptide Toolbox, and succeeded in identifying the most potent SP-protein combinations for three different proteins. The Signal Peptide Toolbox enables an organism-independent, straightforward approach to enhance protein secretion levels.

Evaluation Vector

Peptidosomes offer a perfect platform for enhanced protein overproduction by the means of efficient protein secretion and purification due to physical separation of bacteria and the final product. In order to take full advantage of B. subtilis' native protein secretion potential, it is necessary to evaluate its secretion orchastrating signal peptides. Therefore, we developed the Evaluation Vector. This powerful genetic tool containing a multiple cloning site to easily build all combinatorial constructs of a protein with a set of signal peptides.

Bacillus subtilis

B. subtilis is one of the best-studied gram-positive microorganism, and a model bacterium for studying bacterial differentiation (e.g. endospore formation) and phenotypic heterogeneity. Its ability to become naturally competent makes B. subtilis an organism which is easily accessible for genetic manipulation. The GRAS (generally recognized as safe) status and the secretory capacity made B. subtilis a preferred host of choice for big scale production of secreted proteins, such as lipases, proteases and amylases, highlighting the industrial relevance of this bacterium.