Team:LMU-Munich/Results

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Here we provide you with a quick overview of our key results. Please follow the links to receive more background information.
Here we provide you with a quick overview of our key results. Please follow the links to receive more background information.
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=== Sensing of ''Staphylococcus aureus'' ===
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==[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#sensing Sensing]==
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===Sensing of ''Staphylococcus aureus''===
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Sensing of ''S. aureus'' AIP-II by the two-component system AgrCA [http://parts.igem.org/Part:BBa_K1351036 BBa_K1351036] was evaluated in ''B. subtilis'' using the target promoters P2 [http://parts.igem.org/Part:BBa_K1351037 BBa_K1351037] and P3 [http://parts.igem.org/Part:BBa_K1351037 BBa_K1351037].
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[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#sensing (Further Information)]
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The W168 strain was discovered to possibly be incapable for this experiment due to extracellular proteases destroying the synthetic AIPs. We identified the protease deficient strain ''B. subtilis'' WB700 as proper candicate for the sensing. We successfully transformed the WB700 strain with the sensor construct pBS4S-PxylA-agrCA. Unfortunately, the resistances of this strain were incompatible with our reporter construct.
=== Sensing of ''Streptococcus pneumoniae''===  
=== Sensing of ''Streptococcus pneumoniae''===  
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The two-component system ComDE from ''Streptococcus pneumoniae'', introduced into ''B. subtilis'', can sense the QS peptide CSP and activate the corresponding promoters P<sub>comC</sub> and P<sub>comAB</sub>.
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The two-component system ComDE [http://parts.igem.org/Part:BBa_K1351016 BBa_K1351016]from ''Streptococcus pneumoniae'', introduced into ''B. subtilis'', can sense the QS-peptide CSP and activate the corresponding promoters P<sub>comC</sub> [http://parts.igem.org/Part:BBa_K1351024 BBa_K1351024] and P<sub>comAB</sub> [http://parts.igem.org/Part:BBa_K1351025 BBa_K1351025].
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[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#sensing (Further Information)]
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==[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#adhesion Adhesion]==
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=== Adhesion to ''Streptococcus pneumoniae''===  
=== Adhesion to ''Streptococcus pneumoniae''===  
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Binding of the peptides C4P, CSP and A5P to S. pneumoniae cells was evaluated with the results strongly indicating the adhesion. Our BioBricks [http://parts.igem.org/Part:BBa_K1351001 BBa_K1351001], [http://parts.igem.org/Part:BBa_K1351002 BBa_K1351002] and [http://parts.igem.org/Part:BBa_K1351003 BBa_K1351003] thus can serve as a helpful tool for biomedical applications targeting ''S. pneumoniae''.
Binding of the peptides C4P, CSP and A5P to S. pneumoniae cells was evaluated with the results strongly indicating the adhesion. Our BioBricks [http://parts.igem.org/Part:BBa_K1351001 BBa_K1351001], [http://parts.igem.org/Part:BBa_K1351002 BBa_K1351002] and [http://parts.igem.org/Part:BBa_K1351003 BBa_K1351003] thus can serve as a helpful tool for biomedical applications targeting ''S. pneumoniae''.
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[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#adhesion (Further Information)]
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==[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#killing Killing]==
===Subtilin has inhibitory impact on ''S. pneumoniae'' ===
===Subtilin has inhibitory impact on ''S. pneumoniae'' ===
Spot-on-lawn assays proved the inhibitory effect of the antimicrobial agent on ''S. pneumoniae''.
Spot-on-lawn assays proved the inhibitory effect of the antimicrobial agent on ''S. pneumoniae''.
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[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#killing (Further Information)]
 
===Subtilin has inhibitory impact on ''S. aureus'' ===
===Subtilin has inhibitory impact on ''S. aureus'' ===
Spot-on-lawn assays proved the inhibitory effect of the antimicrobial agent on ''S. aureus'', including MRSA types. Experiments were performed by our collaboration partner [https://2014.igem.org/Team:Groningen iGEM Team Groningen].
Spot-on-lawn assays proved the inhibitory effect of the antimicrobial agent on ''S. aureus'', including MRSA types. Experiments were performed by our collaboration partner [https://2014.igem.org/Team:Groningen iGEM Team Groningen].
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[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#killing (Further Information)]
 
=== SpaIFEG immunity BioBrick ([http://parts.igem.org/Part:BBa_K1351014 BBa K1351014]) mediates self-protection from subtilin.===
=== SpaIFEG immunity BioBrick ([http://parts.igem.org/Part:BBa_K1351014 BBa K1351014]) mediates self-protection from subtilin.===
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These amazing results were evaluated by subtilin-containing supernatant that was filled in holes of agar plates and thus inhibited the growth of the ''B. subtilis'' wild type but inhibited the growth of the ''B. subtilis'' W168 ''amyE''::P<sub>''xyl''</sub>_spaIFEG significantly less.
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These amazing results were evaluated by subtilin-containing supernatant that was filled in holes of agar plates and thus inhibited the growth of the ''B. subtilis'' wild type but inhibited the growth of the ''B. subtilis'' W168 ''amyE''::P<sub>''xyl''</sub>-''spaIFEG'' significantly less.
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=== Suicide Switch===
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A delayed expression can be achieved by wiring the ecf σ factor σ<sub>ecf41</sub>  and its target promoter P<sub>ydfG</sub> between the input promoter and the output. Furthermore, the resistance factor SdpI leads to higher resistance against the antimicrobial peptide SdpC, enabling it to survive until the suicide switch kicks in.
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==[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#suicide Suicide Switch]==
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[https://2014.igem.org/Team:LMU-Munich/Project/Bakillus#suicide (Further Information)]
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=== Ecf41 provides a delayed expression  ===
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=== BioBrickBox===
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A delayed expression can be achieved by wiring the ecf σ factor σ<sub>ecf41</sub>  [http://parts.igem.org/Part:BBa_K823043 BBa_K823043] and its target promoter P<sub>ydfG</sub> [http://parts.igem.org/Part:BBa_K823041 BBa_K823041] between the input promoter and the output.
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=== The SdpI BioBrick can rescue ∆''sdpI B. subtilis'' mutant strains ===
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[https://2014.igem.org/Team:LMU-Munich/Project/BioBrickBox (Further Information)]
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Induceable SdpI [http://parts.igem.org/Part:BBa_K1351017 BBa_K1351017]production leads to higher resistance against the antimicrobial peptide SdpC.
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=== Modeling===
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=== The sdpI-antisense RNA shows no impact regarding accelerated degradation ===  
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We determined the influence of relevant parameters to the dynamic of the induced inhibition of the antitoxin production for the suicide switch. We now can tell which parameters benefit the suicide and which are in reciprocal relation.
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In ''B. subtilis'' W168 cells the expression of the sdpI-antisense RNA did not show any impact on cell vitality.
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[https://2014.igem.org/Team:LMU-Munich/Project/Modeling#modelsuicide (Further Information)]
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Furthermore, we were able to simulate the chemotactical driven movement of BaKillus according to a given concentration gradient of respective substances.
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==[https://2014.igem.org/Team:LMU-Munich/Project/BioBrickBox ''Bacillus'' BioBrick Box]==
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[https://2014.igem.org/Team:LMU-Munich/Project/Modeling (Further Information)]
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To extend the ''Bacillus'' BioBrick Box started by the 2012 team [https://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks LMU-Munich], we created a a few handy parts for ''B. subtilis''.
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=== Application ===  
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==[https://2014.igem.org/Team:LMU-Munich/Project/Modeling#modelsuicide Modeling]==
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We developed a nasal spray for the application of BaKillus against sinusitis.
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We discovered that high translation and transcription rates of the involved mRNA's and proteins play an important role in making the suicide switch work. While high basic concentrations of involved substances are reciprocal to a successful lyse.
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[https://2014.igem.org/Team:LMU-Munich/Application (Further Information)]
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Furthermore, we were able to simulate the chemotactical driven movement of BaKillus according to a given concentration gradient of respective substances. Now we have the prototype for a tool helping us to anticipate the statistical propagation of BaKillus in the patient.
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== Application ==
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We developed a [https://2014.igem.org/Team:LMU-Munich/Application/Diagnosis_Treatment nasal spray] for the application of BaKillus against sinusitis. Furthermore we checked in a [https://2014.igem.org/Team:LMU-Munich/Application/Pretest self-experiment] that ''B.subtilis'' is viable in the nasal mucosa and can survive long enough to conduct its job of killing pathogens.
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Latest revision as of 22:14, 4 December 2014

 

Results

Here we provide you with a quick overview of our key results. Please follow the links to receive more background information.

Sensing

Sensing of Staphylococcus aureus

Sensing of S. aureus AIP-II by the two-component system AgrCA [http://parts.igem.org/Part:BBa_K1351036 BBa_K1351036] was evaluated in B. subtilis using the target promoters P2 [http://parts.igem.org/Part:BBa_K1351037 BBa_K1351037] and P3 [http://parts.igem.org/Part:BBa_K1351037 BBa_K1351037].

The W168 strain was discovered to possibly be incapable for this experiment due to extracellular proteases destroying the synthetic AIPs. We identified the protease deficient strain B. subtilis WB700 as proper candicate for the sensing. We successfully transformed the WB700 strain with the sensor construct pBS4S-PxylA-agrCA. Unfortunately, the resistances of this strain were incompatible with our reporter construct.

Sensing of Streptococcus pneumoniae

The two-component system ComDE [http://parts.igem.org/Part:BBa_K1351016 BBa_K1351016]from Streptococcus pneumoniae, introduced into B. subtilis, can sense the QS-peptide CSP and activate the corresponding promoters PcomC [http://parts.igem.org/Part:BBa_K1351024 BBa_K1351024] and PcomAB [http://parts.igem.org/Part:BBa_K1351025 BBa_K1351025].

Adhesion

Adhesion to Streptococcus pneumoniae

Three peptides were shown to bind to S. pneumoniae when fused to a maltose binding protein. Anchors to the B. subtilis cell wall are still in work.

Binding of the peptides C4P, CSP and A5P to S. pneumoniae cells was evaluated with the results strongly indicating the adhesion. Our BioBricks [http://parts.igem.org/Part:BBa_K1351001 BBa_K1351001], [http://parts.igem.org/Part:BBa_K1351002 BBa_K1351002] and [http://parts.igem.org/Part:BBa_K1351003 BBa_K1351003] thus can serve as a helpful tool for biomedical applications targeting S. pneumoniae.

Killing

Subtilin has inhibitory impact on S. pneumoniae

Spot-on-lawn assays proved the inhibitory effect of the antimicrobial agent on S. pneumoniae.

Subtilin has inhibitory impact on S. aureus

Spot-on-lawn assays proved the inhibitory effect of the antimicrobial agent on S. aureus, including MRSA types. Experiments were performed by our collaboration partner iGEM Team Groningen.

SpaIFEG immunity BioBrick ([http://parts.igem.org/Part:BBa_K1351014 BBa K1351014]) mediates self-protection from subtilin.

These amazing results were evaluated by subtilin-containing supernatant that was filled in holes of agar plates and thus inhibited the growth of the B. subtilis wild type but inhibited the growth of the B. subtilis W168 amyE::Pxyl-spaIFEG significantly less.

Suicide Switch

Ecf41 provides a delayed expression

A delayed expression can be achieved by wiring the ecf σ factor σecf41 [http://parts.igem.org/Part:BBa_K823043 BBa_K823043] and its target promoter PydfG [http://parts.igem.org/Part:BBa_K823041 BBa_K823041] between the input promoter and the output.

The SdpI BioBrick can rescue ∆sdpI B. subtilis mutant strains

Induceable SdpI [http://parts.igem.org/Part:BBa_K1351017 BBa_K1351017]production leads to higher resistance against the antimicrobial peptide SdpC.

The sdpI-antisense RNA shows no impact regarding accelerated degradation

In B. subtilis W168 cells the expression of the sdpI-antisense RNA did not show any impact on cell vitality.

Bacillus BioBrick Box

To extend the Bacillus BioBrick Box started by the 2012 team LMU-Munich, we created a a few handy parts for B. subtilis.

Modeling

We discovered that high translation and transcription rates of the involved mRNA's and proteins play an important role in making the suicide switch work. While high basic concentrations of involved substances are reciprocal to a successful lyse.

Furthermore, we were able to simulate the chemotactical driven movement of BaKillus according to a given concentration gradient of respective substances. Now we have the prototype for a tool helping us to anticipate the statistical propagation of BaKillus in the patient.

Application

We developed a nasal spray for the application of BaKillus against sinusitis. Furthermore we checked in a self-experiment that B.subtilis is viable in the nasal mucosa and can survive long enough to conduct its job of killing pathogens.

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.

Retrieved from "http://2014.igem.org/Team:LMU-Munich/Results"