Team:LMU-Munich/Bacillus Introduction

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Bacillus subtilis - a new chassis for iGEM

We chose to work with Bacillus subtilis to set new horizons in the Escherichia coli-dominated world of iGEM by offering a set of tool for this gram-positive model organism to the . To introduce B. subtilis, we first want to highlight some important aspects of this organism, which are listed in the table below.

In general, bacteria can be divided into two major groups that differ primarily in their cell envelope: gram-positive and gram-negative. E. coli is the model organism for the gram-negative bacteria. A model organism for the gram-positive bacteria is B. subtilis, our favourite pet. The natural habitat of B. subtilis is the soil, so it is forced to adapt to numerous environmental changes. Accordingly, B. subtilis is characterized by quick and cunning reflexes and a complex lifestyle. There are many differentiations and survival strategies that B. subtilis can engage (Fig. 1): Due to its natural competence, it can take up exogenous DNA and integrate it into its genome. To be flexibel to the environment and move towards nutrients or avoid toxic compounds, it is motile with the aid of its peritrichous flagella. If starved some cells even become cannibals that feast on their siblings. If the conditions get too bad for survival, B. subtilis can form endospores. These are very dormant and highly stable stages that are resistent against e.g. desiccation, UV light, heat and pressure. Once these spores encounter better conditions they are able to germinate again. See this section for Details on germination.

Fig. 1: Schematic representation of the distinct cell types that differentiate in the communities of Bacillus subtilis(taken from Lopez & Kolter, 2010).

There are two major differences between B. subtilis and E. coli that are of interest to us:

1) Transformation of B. subtilis
As B. subtilis and E. coli are model organisms, they have established genetics. The advantage of B. subtilis is that it is naturally competent. So it is very easy to conduct genetic manipulations. It can replicate plasmids as E. coli does, but there is a much more elegant way of bringing in exogenous DNA fragments. When flanked by regions homologous to the B. subtilis genome, it will be integrated at high efficiency via homologous recombination at this locus and subsequently be replicated with the chromosome (Fig. 2). This leads to stable, single-copy genomic alterations. Thereby avoiding, copy-number artifacts occuring with replicative plasmids. This different way of genetic manipulations requires the use of integrative vectors as provided by our BacillusBioBrickBox. For this reason, B. subtilis is an ideal genetic platform for Synthetic Bioloy. But so far, very few iGEM teams have worked with this model organism due to the lack of suitable BioBrick-compatible genetic tools.

Fig. 2: Exogenous DNA is shown in red while the bacterial genome is black a) The propagation of exogenous DNA if brought in as replicative plasmid. The number of plasmids per cell can vary. b) The propagation of exogenous DNA if it is able to integrate into the genome via homologous recombination

2) Differentiation

B. subtilis is able to differentiate into cells with different morphologies and functions (Fig. 1 and Fig. 3). The most characteristic form is the endospore, which is produced under nutrient starvation. In our project, we will exploit the production of endospores. Because they are extremely stable, they are perfect vehicles for the display of functional fusion proteins on their surface as illustrated by our Sporobead module.

Fig. 3: The vegetative cycle is very similiar to the one of E. coli. But if there is a stress like for example starvation, the cells enter sporulation, where they first undergo a polar cell division, followed by the formation of the endospore. If the enviromental conditions are suitable again, the spore will then germinate and reenter the vegetative cycle.

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