Team:LMU-Munich/Bacillus Introduction

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The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".

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Bacillus subtilis - a new chassis for iGEM

We chose to work with Bacillus subtilis to set new horizons and offer tools for this model organism to the Escherichia coli-dominated world of iGEM. In addition, B. subtilis produces spores which we use for our Sporobeads. To introduce B. subtilis to the iGEM world, we want to present some important aspects of this organism, as they differ from the commonly used Escherichia coli.

Organism	B. subtilis	E. coli
Type	gram-positive rod	gram-negative rod
Motility	+	+
Transformation	natural competence	artificial competence
Vectors	integrative & replicative	replicative
Sporulation	+	-
Differentiation	+	-
Safety	GRAS	GRAS

In contrast to E. coli, the model organism B. subtilis is a gram-postivite rod. It is a facultatively aerobic soil bacterium which can move with its peritrichous flagella. Under ideal conditions, it has a doubling time of 45 minutes. B. subtilis is, in contrast to E. coli, naturally competent, which means that e.g. under nutrient limitation, 10% of cells in a populaton get competent and take up DNA. Also unique for B. subtilis is that the vegetative cells can differentiate under nutrient limitations into spores, which we use in our project Beadzillus. Concerning working with B. subtilis, it is important to know that there are integrative vectors which can integrate into the genome of the organism, in contrast to E. coli, for which there are only replicative vectors.

Visit our Protocols page for details how to work with B. subtilis

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

1) B. subtilis is able to differentiate into cells with different morphology and function (Fig. 1), the most severe form being the endospore which is produced under stress conditions. But there are also phenomena like cannibalism which makes B. subtilis a lot more diverse to work with. We will exploit the production of endospores in our project Beadzillus. The life cycle of B. subtilis is depicted in Fig. 2.

Fig. 1:

Fig. 2: The vegetative cycle is very similiar to the one of E. coli. But if there is a stress condition like 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.

2) B. subtilis can replicate exogenous DNA via an origin of replication on a plasmid as E. coli does, but there is a much more elegant way of bringing in exogenous DNA stretches. When flanked by homologous regions to the bacterial genome, it will integrate at high efficiency via homologous recombination at this locus and furthermore be replicated with the genome. This has the advantage that if comparing different variables, not only the enviroment is always the same, but also the copy number is from cell to cell and from strain to strain the same, which is not always the case for replicative plasmids. This integrative way of bringing in exogenous DNA was exploited by us when producing the BioBrick compatible Bacillus vectors. The comparision between these two ways of bringing in exogenous DNA is depicted in Fig. 3.
For these reasons in some cases B. subtilis can be the chassis of choice. Sadly very few teams have worked with this model organism and there is at this time no established BioBrick system to use B. subtilis as a chassis.

Fig. 3: Exogenic DNA is shown in red while the bacterial genome is black a) The propagation of exogenic DNA if brought in as replicative plasmid. The number of plasmids per cell can vary. b) The propagation of exogenic DNA if it is able to integrate into the genome via homologous recombination

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Bacillus Intro	Bacillus BioBrickBox	Sporobeads	Germination STOP

Retrieved from "http://2012.igem.org/Team:LMU-Munich/Bacillus_Introduction"

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-'''1)''' ''B. subtilis'' is able to differentiate into cells with different morphology and function ('''Fig. 1'''), the most severe form being the endospore which is formed under stress conditions. But there are also phenomena like cannibalism which makes ''B. subtilis'' a lot more diverse to work with. We will exploit the production of endospores in our project '''Bead'''zillus. The life cycle of ''B. subtilis'' is depicted in '''Fig. 2'''.
+'''1)''' ''B. subtilis'' is able to differentiate into cells with different morphology and function ('''Fig. 1'''), the most severe form being the endospore which is produced under stress conditions. But there are also phenomena like cannibalism which makes ''B. subtilis'' a lot more diverse to work with. We will exploit the production of endospores in our project '''Bead'''zillus. The life cycle of ''B. subtilis'' is depicted in Fig. 2.
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-'''2)''' ''B. subtilis'' can replicate exogenic DNA via an origin of replication on a plasmid as ''E. coli'' does, but there is a much more elegant way of bringing in exogenic DNA stretches. When flanked by homologous regions to the bacterial genome, it will integrate at high efficiency via homologous recombination at this locus and furthermore be replicated with the genome. This has the advantage that if comparing different things, not only the enviroment is always the same, but also the copy number is from cell to cell and from strain to strain the same, which is not always the case for replicative plasmids. This integrative way of bringing in exogenic DNA will be exploited by us when producing the BioBrick compatible ''Bacillus'' vectors. The comparision between these two ways of bringing in exogenic DNA  is depicted in Fig. 3.
+'''2)''' ''B. subtilis'' can replicate exogenous DNA via an origin of replication on a plasmid as ''E. coli'' does, but there is a much more elegant way of bringing in exogenous DNA stretches. When flanked by homologous regions to the bacterial genome, it will integrate at high efficiency via homologous recombination at this locus and furthermore be replicated with the genome. This has the advantage that if comparing different variables, not only the enviroment is always the same, but also the copy number is from cell to cell and from strain to strain the same, which is not always the case for replicative plasmids. This integrative way of bringing in exogenous DNA was exploited by us when producing the BioBrick compatible ''Bacillus'' vectors. The comparision between these two ways of bringing in exogenous DNA  is depicted in Fig. 3.
 <br>
 For these reasons in some cases ''B. subtilis'' can be the chassis of choice. Sadly very few teams have worked with this model organism and there is at this time no established BioBrick system to use ''B. subtilis'' as a chassis.