Team:LMU-Munich/Project/B subtilis

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Revision as of 19:29, 13 October 2014

TOUR

Bacillus subtilis

We aim to introduce quorum sensing modules from the two Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae into an appropriate chassis that will sense and eventually kill these pathogens specifically.

As Bacillus subtilis is a Gram-positive rod shaped bacterium and many of us had good experiences working with this microorganism, we took B. subtilis as our chassis of choice. Furthermore, the guanine-cytosine contents of B. subtilis (43.5 %), S. aureus (32.8 %) and S. pneumoniae (39.7 %) are comparable, which is of great advantage for heterologous gene expression.

Fig. 1: Schematic representation of the different cell fates that coexist in a B. subtilis population (taken from Lopez & Kolter, 2010 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Extracellular+signals+that+define+distinct+and+coexisting+cell+fates+in+Bacillus+subtilis]).

B. subtilis is a soil bacterium that has to face changing environmental stimuli like quorum-sensing molecules, temperature, pH or scarcity of nutrients. In order to cope with this, B. subtilis is able to differentiate into different cell types. [http://www.ncbi.nlm.nih.gov/pubmed/20030732] During starvation, B. subtilis is able to kill his siblings to gain new nutrition, introducing specific toxins to the extraplasmic space. If the conditions get even worse, the soil bacterium is able to form long living, stable endospores. However, if nutrients become available again, they are able to germinate and reenter the vegetative cycle.

Furthermore, our model organism is a nonpathogenic and nontoxicogenic strain and was declared as GRAS (Generally Regarded As Safe) by the Food and Drug Administration [http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/MicroorganismsMicrobialDerivedIngredients/default.htm (FDA)].

In addition, the iGEM Team LMU-Munich 2012 has constructed the Bacillus BioBrickBox (B4), which contains several well evaluated integrative and replicative vectors for B. subtilis [1], thus providing a powerful toolbox for the engineering of B. subtilis. One extraordinary aspect of B. subtilis is its ability to perform natural transformation in its postexponential phase of cell growth. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC372826/]. This feature can be used in the lab very reliably. (Link zu Protokollsammlung B.subtilis Trafo). For example, integrative B4 vectors are constructed so that the DNA fragment of interst is flanked by homologous regions to the B. subtilis genome. Thus, via homologous recombination at this locus, B. subtilis will integrate and replicate the inserted DNA fragment.

Due to all of these helpful features, B. subtilis is our chassis organism of choice! Check out our results, obtained with this organism!

Hi there!

Welcome to our Wiki! I'm BaKillus, the pathogen-hunting microbe, and I'll guide you on this tour through our project. If you want to learn more about a specific step, you can simply close the tour and come back to it anytime you like. So let's start!

What's the problem?

First of all, what am I doing here? The problem is, pathogenic bacteria all around the world are becoming more and more resistant against antimicrobial drugs. One major reason for the trend is the inappropriate use of drugs. With my BaKillus super powers, I want to reduce this misuse and thus do my part to save global health.

Sensing of pathogens

To combat the pathogenic bacteria, I simply eavesdrop on their communication. Bacteria talk with each other via quorum sensing systems, which I use to detect them and trigger my responses.

Adhesion

The more specific and effective I can use my powers, the lower the danger is of provoking new resistance development. So I catch pathogens whenever I get hold of them and stick to them until my work is done.

Killing

Talking about my work - killing pathogens is finally what I am made for. In response to quorum sensing molecules of the pathogens, I export a range of antimicrobial substances leading to dissipation of biofilms and the killing of the targeted bacteria.

Suicide switch

When the job is done and all the bad guys are finished, you don't need a super hero anymore. So after fulfilling my work I say goodbye to the world by activating my suicide switch.

Application

Of course I'm not only a fictional hero, but a very real one. In two different prototypes, I could be used for diagnosis or treatment of pathogen-caused diseases. However, there is still a whole lot of regulational and economical questions that have to be answered before.

See you!

So now you know my short story - and it is time for me to return to my fight for a safer world. Feel free to take a closer look on my super powers, the process of my development or the plans for a medical application.

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 As ''Bacillus subtilis'' is a Gram-positive rod shaped bacterium and many of us had good experiences working with this microorganism, we took ''B. subtilis'' as our chassis of choice. Furthermore, the guanine-cytosine contents of ''B. subtilis'' (43.5 %), ''S. aureus'' (32.8 %) and ''S. pneumoniae'' (39.7 %) are comparable, which is of great advantage for heterologous gene expression.
-[[File:LMU14 Cell fates Bsubtilis.png|Fig. 1: Schematic representation of the different cell fates that coexist in a ''B. subtilis'' population (taken from Lopez & Kolter, 2010 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Extracellular+signals+that+define+distinct+and+coexisting+cell+fates+in+Bacillus+subtilis]).]]
+[[File:LMU14 Cell fates Bsubtilis.png|left|frame|Fig. 1: Schematic representation of the different cell fates that coexist in a ''B. subtilis'' population (taken from Lopez & Kolter, 2010 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Extracellular+signals+that+define+distinct+and+coexisting+cell+fates+in+Bacillus+subtilis]).]]
 ''B. subtilis'' is a soil bacterium that has to face changing environmental stimuli like quorum-sensing molecules, temperature, pH or scarcity of nutrients. In order to cope with this, ''B. subtilis'' is able to differentiate into different cell types. [http://www.ncbi.nlm.nih.gov/pubmed/20030732] During starvation, ''B. subtilis'' is able to kill his siblings to gain new nutrition, introducing specific toxins to the extraplasmic space. If the conditions get even worse, the soil bacterium is able to form long living, stable endospores. However, if nutrients become available again, they are able to germinate and reenter the vegetative cycle.