Difference between revisions of "Team:Groningen/Experiments"

 
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<p>The biological realization of CryptoGErM consisted of four  
 
<p>The biological realization of CryptoGErM consisted of four  
−
subprojects: integration, characterization, key hiding and key deletion. The encrypted message and the key were integrated into the genome of <em>B. subtilis</em>. The location in the genomic DNA makes it harder to retrieve the  
+
subprojects: integration, characterization, key hiding and key  
−
data, since whole genome sequencing is required. The message is  
+
deletion. The encrypted message and the key were integrated into  
−
protected by a digital lock: the key, and thus doesn’t need further  
+
the genome of <em>B. subtilis</em>. The location in the genomic DNA  
−
biological protection. The key is not encrypted and has to be protected by a biological  
+
makes it harder to retrieve the data, since whole genome sequencing  
−
lock. We followed different approaches to design a multi-layered  
+
is required. The message is protected by a digital lock: the key,  
−
biological lock. We followed two main ideas, namely hiding the key and deleting  
+
and thus doesn’t need further biological protection. The key is not  
−
the key when unauthorized parties handle it. This system is highly flexible and biological layers can easily  
+
encrypted and has to be protected by a biological lock. We followed  
−
be added or modified to the wishes of the user.</p>
+
different approaches to design a multi-layered biological lock. We  
 +
followed two main ideas, namely hiding the key and deleting the key  
 +
when unauthorized parties handle it. This system is highly flexible  
 +
and biological layers can easily be added or modified to the wishes  
 +
of the user. An overview of all the protocols and plasmid construction can be found in the <a href="/Team:Groningen/Labjournal">Lab journal</a>.</p>
 
 
 
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<div class="split">
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message and key transmission these <em>B. subtilis</em> strains were  
 
message and key transmission these <em>B. subtilis</em> strains were  
 
sporulated. We were able to successfully retrieve and read out our message. </p>
 
sporulated. We were able to successfully retrieve and read out our message. </p>
−
<p><ul><li><a href="/Team:Groningen/Proof">Read more about the integration and our proof of concept.</a></ul></li></p>
 
 
 
 +
<p><ul>
 +
<li><a href="/Team:Groningen/Proof">Read more about the integration and our proof of concept.</a></li>
 +
</ul></p>
 
</div>
 
</div>
 
<div class="right flone">
 
<div class="right flone">
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<img src="https://static.igem.org/mediawiki/2016/4/40/T--Groningen--layersguy.png" />
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<img src="https://static.igem.org/mediawiki/2016/a/a1/T--Groningen--Bacillus-couple.png" />
 
</div>
 
</div>
 
</div>
 
</div>
  
−
+
<div class="split">
−
 
+
−
 
+
−
+
−
                     
+
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<div class="split">
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<div class="right fltwo">
 
<div class="right fltwo">
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<h2>Characterization</h2>
+
<h2>Characterization</h2>
 +
 +
<p>The integration vector from team LMU Munich
 +
BacillusBiobrickbox 2012 (<a
 +
href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>)
 +
can be used to integrate an insert into the amyE locus of
 +
<em>B. subtilis</em>. In order to be able to efficiently
 +
use this integration vector we characterized BBa_K823023 by
 +
determining its transformation efficiency. This helped us
 +
in our project and will hopefully help future iGEM teams as
 +
well. </p>
  
−
                        <p>The integration vector from team LMU Munich BacillusBiobrickbox 2012 (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>)
+
<p><ul>
−
can be used to integrate an insert into the amyE locus of <em>B. subtilis</em>. In order to
+
<li><a href="/Team:Groningen/BrickCharacter">Read more about how we determined the transformation efficiency..</a></ul></li>
−
be able to efficiently use this integration vector we characterized BBa_K823023 by determining its
+
</p></ul>
−
transformation efficiency. This helped us in our project and will hopefully help future iGEM teams aswell.
+
</div>
−
</p>
+
−
 
+
−
<p><ul><li><a href="/Team:Groningen/BrickCharacter">Read more about how we determined the transformation efficiency..</a></ul></li></p>
+
−
 
+
−
</div>
+
 
<div class="left flone">
 
<div class="left flone">
 
<img src="https://static.igem.org/mediawiki/2016/8/86/T--Groningen--TransformEffK8-4.jpg" />
 
<img src="https://static.igem.org/mediawiki/2016/8/86/T--Groningen--TransformEffK8-4.jpg" />
 
</div>
 
</div>
−
 
+
</div>
−
</div>                      
+
−
<div class="split">
+
<div class="split">
 
<div class="left fltwo">
 
<div class="left fltwo">
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<h2>Key hiding</h2>
+
<h2>Key hiding</h2>
  
−
                     
+
<h4>Decoy</h4>
  
−
<h4>Decoy</h4>
+
<p>Hiding the key became called the <em>decoy</em>
 +
approach.The key-containing spore will be send in a mixture
 +
of different decoy spores. The recipient has to be aware of
 +
the special treatment that is required to select the
 +
correct spores from the decoy. We have been working on a
 +
photoswitchable ciprofloxacin compound. </p>
  
−
+
<p>Only the knowledge about the right wavelength allows the  
−
<p>Hiding the key became called the <em>decoy</em> approach.The
+
recipient to activate the added ciprofloxacin and thus
−
key-containing spore will be send in a mixture of different decoy
+
start selection of the right spore strain.</p>
−
spores. The recipient has to be aware of the special treatment that
+
−
is required to select the correct spores from the decoy. We have
+
−
been working on a photoswitchable ciprofloxacin compound. </p>
+
  
−
<p>Only the knowledge about the right wavelength allows the
+
<p>Our design of the decoy approach included the biobricks  
−
recipient to activate the added ciprofloxacin and thus start
+
for ciprofloxacin resistance, a superfolder GFP and a pATP  
−
selection of the right spore strain.</p>
+
promotor.</p>
−
 
+
−
<p>Our design of the decoy approach included the biobricks for  
+
<p><ul>
−
ciprofloxacin resistance, a superfolder GFP and a pATP promotor.</p>
+
<li><a href="/Team:Groningen/Decoy">Read more about the decoy experiment.</a></ul></li>
−
<p><ul><li><a href="/Team:Groningen/Decoy">Read more about the decoy experiment.</a></ul></li></p>
+
</u></p>
−
 
+
</div>
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<h4>MIC and MBC values of ciprofloxacin</h4>
+
−
                        <p>We determined the MIC and MBC of ciprofloxacin on wild-type <em>Bacillus subtilis</em> 168 as well as the MIC of <em>E. coli</em> Top 10
+
−
and <em>B. subtilis</em> 168 carrying the qnrS1 ciprofloxacin resistance gene. We could observe  a significant
+
−
improvement in antibiotic tolerance when compared to the MIC values of the wild-type
+
−
strains Additionally we obtained a <em>B. subtilis</em> 168 isolate by directed evolution which is even more resistant to ciprofloxacin.
+
−
</p>
+
−
<p><ul><li><a href="/Team:Groningen/PhotoswitchableAntibiotics">Read more about the MIC and MBC experiments..</a></ul></li></p>
+
−
</div>
+
 
<div class="right flone">
 
<div class="right flone">
 
<img src="https://static.igem.org/mediawiki/2016/d/d3/T--Groningen--Decoy-4_2.jpg" />
 
<img src="https://static.igem.org/mediawiki/2016/d/d3/T--Groningen--Decoy-4_2.jpg" />
 
</div>
 
</div>
 +
</div>
 +
<div class="split">
 +
<div class="left fltwo">
 +
<h4>MIC and MBC values of ciprofloxacin</h4>
 +
                       
 +
<p>We determined the MIC and MBC of ciprofloxacin on
 +
wild-type <em>Bacillus subtilis</em> 168 as well as the MIC
 +
of <em>E. coli</em> Top 10 and <em>B. subtilis</em> 168
 +
carrying the <em>qnrS1</em> ciprofloxacin resistance gene.
 +
We could observe  a significant improvement in antibiotic
 +
tolerance when compared to the MIC values of the wild-type
 +
strains. Additionally we obtained a <em>B. subtilis</em>
 +
168 isolate by directed evolution which is even more
 +
resistant to ciprofloxacin. </p>
 +
 +
<small>Our time-lapse video to the right shows germination of <em>B. subtilis</em></small>
  
−
 
+
<p><ul>
−
</div>
+
<li><a href="/Team:Groningen/PhotoswitchableAntibiotics">Read more about the MIC and MBC experiments..</a></ul></li>
−
<div class="split">
+
</ul></p>
 +
</div>
 +
<div class="right flone">
 +
<video controls preload="metadata">
 +
<source src="https://static.igem.org/mediawiki/2016/1/11/T--Groningen--Spore-Movie.mp4" />
 +
</video>
 +
</div>
 +
</div>
 +
 +
<div class="split">
 
<div class="right fltwo">
 
<div class="right fltwo">
−
<h2>Key deletion</h2>
+
<h2>Key deletion</h2>
  
−
<h4>NucA key deletion</h4>
+
<h4>NucA key deletion</h4>
 
 
−
<p>Another biological security layer is provided by our key  
+
<p>Another biological security layer is provided by our key  
−
deletion system. This assures that only authorized parties can access the key. We have been working on two different approaches.  
+
deletion system. This assures that only authorized parties  
−
The first is a nucA killswitch which is made out of an assembly of  
+
can access the key. We have been working on two different  
−
different BioBricks. Atc or tetracycline have to be added to  
+
approaches. The first is a nucA killswitch which is made  
−
inhibit the tetR promoter to stop the expression of the nucA and  
+
out of an assembly of different BioBricks. ATc or  
−
digestion of the key sequence.</p>
+
tetracycline have to be added to inhibit the tetR promoter  
 +
to stop the expression of the nucA and digestion of the key  
 +
sequence.</p>
  
−
 
+
<h4>CRISPR/Cas9 key deletion</h4>
−
 
+
−
 
+
−
<h4>CRISPR/Cas9 key deletion</h4>
+
 
 
−
<p>The second approach makes use of a CRISPR/Cas9 system which will  
+
<p>The second approach makes use of a CRISPR/Cas9 system  
−
delete the key from the genome if no special treatment is applied.  
+
which will delete the key from the genome if no special  
−
Addition of aTc or tetracycline will stop the Cas9 expression.</p>
+
treatment is applied. Addition of aTc or tetracycline will  
 +
stop the Cas9 expression.</p>
  
−
<p><ul><li><a href="/Team:Groningen/KeyDeletion">Read more about the key deletion.</a></ul></li></p>
+
<p><ul>
−
 
+
<li><a href="/Team:Groningen/KeyDeletion">Read more about the key deletion.</a></ul></li>
−
 
+
</ul></p>
−
</div>
+
</div>
 
<div class="left flone">
 
<div class="left flone">
 
<img src="https://static.igem.org/mediawiki/2016/4/4f/T-Groningen-nucA-in-pSB1C3-plate2.jpg" />
 
<img src="https://static.igem.org/mediawiki/2016/4/4f/T-Groningen-nucA-in-pSB1C3-plate2.jpg" />
 
</div>
 
</div>
−
 
+
</div>
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</div>  
+
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+
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</section>
 
</section>
 
</article>
 
</article>
 
</html>
 
</html>
 
{{Groningen/footer}}
 
{{Groningen/footer}}

Latest revision as of 22:15, 19 October 2016

CryptoGE®M
CryptoGE®M
Team
Team
Members Supervisors Collaborations Achievements Contact
Project
Project
Tour Description Design Results Demonstrate Applications Future Notebook
Biology
Biology
Experiments Parts Safety Lab journal
Computing
Computing
Modelling Software Encoding Decoding
Human Practice
Human Practices
Human Practices Gold Silver
Acknowledgements
Support
Sponsors Attributions

Experiments

The biological realization of CryptoGErM consisted of four subprojects: integration, characterization, key hiding and key deletion. The encrypted message and the key were integrated into the genome of B. subtilis. The location in the genomic DNA makes it harder to retrieve the data, since whole genome sequencing is required. The message is protected by a digital lock: the key, and thus doesn’t need further biological protection. The key is not encrypted and has to be protected by a biological lock. We followed different approaches to design a multi-layered biological lock. We followed two main ideas, namely hiding the key and deleting the key when unauthorized parties handle it. This system is highly flexible and biological layers can easily be added or modified to the wishes of the user. An overview of all the protocols and plasmid construction can be found in the Lab journal.

Integration

The first subproject is the integration, the key and message sequences were integrated into the genomic DNA of two separate Bacillus subtilis strains. In order to achieve this we had the key and the message sequence synthesized by IDT. Then we cloned it in pSB1C3 to amplify it from there and cloned it in the B. subtilis integration biobrick BBa_K823023. This vector can be used to integrate sequences into the amyE locus of Bacillus subtilis. From there we integrated it in the genome of B. subtilis 168 trp+. For message and key transmission these B. subtilis strains were sporulated. We were able to successfully retrieve and read out our message.

Characterization

The integration vector from team LMU Munich BacillusBiobrickbox 2012 (BBa_K823023) can be used to integrate an insert into the amyE locus of B. subtilis. In order to be able to efficiently use this integration vector we characterized BBa_K823023 by determining its transformation efficiency. This helped us in our project and will hopefully help future iGEM teams as well.

Key hiding

Decoy

Hiding the key became called the decoy approach.The key-containing spore will be send in a mixture of different decoy spores. The recipient has to be aware of the special treatment that is required to select the correct spores from the decoy. We have been working on a photoswitchable ciprofloxacin compound.

Only the knowledge about the right wavelength allows the recipient to activate the added ciprofloxacin and thus start selection of the right spore strain.

Our design of the decoy approach included the biobricks for ciprofloxacin resistance, a superfolder GFP and a pATP promotor.

MIC and MBC values of ciprofloxacin

We determined the MIC and MBC of ciprofloxacin on wild-type Bacillus subtilis 168 as well as the MIC of E. coli Top 10 and B. subtilis 168 carrying the qnrS1 ciprofloxacin resistance gene. We could observe a significant improvement in antibiotic tolerance when compared to the MIC values of the wild-type strains. Additionally we obtained a B. subtilis 168 isolate by directed evolution which is even more resistant to ciprofloxacin.

Our time-lapse video to the right shows germination of B. subtilis

Key deletion

NucA key deletion

Another biological security layer is provided by our key deletion system. This assures that only authorized parties can access the key. We have been working on two different approaches. The first is a nucA killswitch which is made out of an assembly of different BioBricks. ATc or tetracycline have to be added to inhibit the tetR promoter to stop the expression of the nucA and digestion of the key sequence.

CRISPR/Cas9 key deletion

The second approach makes use of a CRISPR/Cas9 system which will delete the key from the genome if no special treatment is applied. Addition of aTc or tetracycline will stop the Cas9 expression.

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Team
Project
Biology
Modelling
Computing
Human Practices
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