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.

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, whole-cell biosensors constitute a helpful investigative tool. In this subproject, we developed a functional and complete heterologous Beta-lactam biosensor in Bacillus subtilis. By the time these specified cells sense a compound of the beta-lactam family, they will respond by producing a measurable luminescence signal. Thereby, we analyzed 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 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 proofing 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

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 partsregistry) enables an organism-independent, straightforward approach to identifying the best combination of SP with any protein of interest

Evaluation Vector

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.

Bacillus subtilis – The gram-positive model organism

B. subtilis is the best-studied gram-positive microorganism, and a model bacterium for studying bacterial differentiation (e.g. endospore formation) and phenotypic heterogeneity.[1][2] Its ability to become naturally competent makes B. subtilis an organism with easily tractable genetics.[3] The GRAS (generally recognized as safe) status and 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. [5]

In addition, the iGEM Team LMU-Munich 2012 has constructed the Bacillus BioBrickBox, which contains several well evaluated integrative vectors and other parts for the use in B. subtilis, thus providing a powerful toolbox to engineer B. subtilis.[6]

References

[1] Lopez, D., Vlamakis, H. & Kolter, R. (2009) Generation of multiple cell types in Bacillus subtilis: from soil bacterium to super-secreting cell factory. FEMS Microbiol. Rev., 33, 152–163.
[2] Lopez, D. & Kolter, R. (2010) Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis. FEMS Microbiol. Rev. 34, 134–149
[3] Kaufenstein, M., van der Laan, M. & Graumann, P. L. (2011) The three-layered DNA uptake machinery at the cell pole in competent Bacillus subtilis cells is a stable complex. J. Bacteriol. 193, 1633–1642.
[4] Fu L. L., Xu Z. R., Li W. F., Shuai J. B., Lu P. and Hu C. X. (2006) Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnology advances 25, 1 (1-12).
[5] Harwood, C. R. (1992) Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol. 10, 247–256
[6] Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., Kelterborn, S., Sauer, C., Fritz, G., Gebhard, S., and Mascher, T. (2013) The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7, 29.