Team:LMU-Munich/Bacillus Introduction

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==''Bacillus subtilis'' - a new chassis for iGEM==
==''Bacillus subtilis'' - a new chassis for iGEM==
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<br>
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<p align="justify">We chose to work with ''Bacillus subtilis'' to set new horizons in the ''Escherichia coli''-dominated world of iGEM by offering a set of tools for this Gram-positive model organism to the public domain. To introduce ''B. subtilis'', we first want to highlight some important aspects of this organism, which are listed in the table below.</p>
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====Introduction====
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{| class="colored" width="100%" style="text-align:center;"
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|<p align="justify">Introduction to ''B. subtilis'' and background information.</p>
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|[[File:Tabelle.png|right|150px|link=Team:LMU-Munich/Data/introintro]]
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!Organism
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!''B. subtilis''
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!''E. coli''
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!<font color="#EBFCE4">Type</font>
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|Gram-positive rod
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|Gram-negative rod
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!Motility
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!Transformation
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|natural competence
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|artificial competence
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!Vectors
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|integrative & replicative
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|replicative
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!Sporulation
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!Differentiation
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!Safety
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|[http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm GRAS]
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|[http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/default.htm GRAS]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/introintro]]
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<p align="justify"> 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 flexible to the environment and move towards nutrients or avoid toxic agents, 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 resistant against e.g. desiccation, UV light, heat and pressure. Once these spores encounter better conditions they are able to germinate again. See this [https://2012.igem.org/Team:LMU-Munich/Germination_Stop section] for Details on germination.</p>
 
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There are two major differences between ''B. subtilis'' and ''E. coli'' that are of interest to us:
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| style="width: 70%;background-color: #EBFCE4;" |
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====Transformation of ''B. subtilis''====
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{|
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{| width="100%" style="text-align:center;"|
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|[[File:LMU Bacillus life.png|center|450px]]
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|<p align="justify">Cloning strategy for the work of '' B. subtilis''.</p>
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|[[File:Integration.png|right|150px|link=Team:LMU-Munich/Data/integration]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/integration]]
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<font color="#000000"; size="2"><p align="justify">Fig. 1: Schematic representation of the distinct cell types that differentiate in the communities of ''Bacillus subtilis''(taken from [http://www.ncbi.nlm.nih.gov/pubmed/20030732 Lopez & Kolter, 2010]).</p></font>
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====Differentiation====
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{| width="100%" style="text-align:center;"|
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There are two major differences between ''B. subtilis'' and ''E. coli'' that are of interest to us:
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|<p align="justify">The life cycle of ''B. subtilis'' and the different cell stages.</p>
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|[[File:Figures Bacillus Intro fig1.png|right|150px|link=Team:LMU-Munich/Data/differentiation]]
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'''1) Transformation of ''B. subtilis'''''
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<p align="justify">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 [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks 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.</p>
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|[[File:LMU-Munich-insertion-Intro1.png|500px|center]]
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! colspan="2" |[[File:LMU Arrow purple.png|40px|link=Team:LMU-Munich/Data/differentiation]]
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|[[File:LMU-Munich-insertion-Intro2.png|500px|center]]
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<font color="#000000"; size="2"><p align="justify">Fig. 2: Uptake and integration of linear exogenous DNA into the genome via homologous recombination.</p></font>
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'''2) Differentiation'''
 
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<p align="justify">''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 [https://2012.igem.org/Team:LMU-Munich/Spore_Coat_Proteins '''Sporo'''bead] module.
 
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<font color="#000000"; size="2"><p align="justify">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.</p></font>
 
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====Project Navigation====
====Project Navigation====
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Latest revision as of 16:53, 26 October 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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