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− | collapsers.allow.push('proof-of-concept');
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− | collapsers.allow.push('labjournal');
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− | collapsers.allow.push('decoy');
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− | collapsers.allow.push('brick-character');
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− | </script>
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| <!-- Spore movie: https://static.igem.org/mediawiki/2016/1/11/T--Groningen--Spore-Movie.mp4 --> | | <!-- Spore movie: https://static.igem.org/mediawiki/2016/1/11/T--Groningen--Spore-Movie.mp4 --> |
| <article> | | <article> |
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| <h2>Experiments</h2> | | <h2>Experiments</h2> |
| | | |
− | <p>The biological realization of CryptoGErM consisted of three | + | <p>The biological realization of CryptoGErM consisted of four |
− | subprojects: integration, decoy and key deletion.</p> | + | 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 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 <a href="/Team:Groningen/Labjournal">Lab journal</a>.</p> |
| | | |
| <div class="split"> | | <div class="split"> |
| <div class="left flone"> | | <div class="left flone"> |
− | <h4>Integration</h4> | + | <h2>Integration</h2> |
| | | |
| <p>The first subproject is the <em>integration</em>, the key and message | | <p>The first subproject is the <em>integration</em>, the key and message |
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| <em>Bacillus subtilis</em> strains. In order to achieve this we had the key | | <em>Bacillus subtilis</em> strains. In order to achieve this we had the key |
| and the message sequence synthesized by IDT. Then we cloned it in | | 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 | + | pSB1C3 to amplify it from there and cloned it in the <em>B. subtilis</em> |
| integration biobrick <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>. This vector can be used to | | integration biobrick <a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>. This vector can be used to |
| integrate sequences into the amyE locus of <em>Bacillus subtilis</em>. From | | integrate sequences into the amyE locus of <em>Bacillus subtilis</em>. From |
− | there we integrated it in the genome of B. subtilis 168 trp+. For | + | there we integrated it in the genome of <em>B. subtilis</em> 168 trp+. For |
− | message and key transmission these B. subtilis strains were | + | message and key transmission these <em>B. subtilis</em> strains were |
− | sporulated. </p> | + | sporulated. We were able to successfully retrieve and read out our message. </p> |
| | | |
− | <p>The location in the genomic DNA makes it harder to retrieve the | + | <p><ul> |
− | data, since whole genome sequencing is required. The message is
| + | <li><a href="/Team:Groningen/Proof">Read more about the integration and our proof of concept.</a></li> |
− | protected by a digital lock: the key, and thus doesn’t need further
| + | </ul></p> |
− | biological protection. </p>
| + | |
− | | + | |
− | <p>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.</p>
| + | |
| </div> | | </div> |
| <div class="right flone"> | | <div class="right flone"> |
− | <img src="https://static.igem.org/mediawiki/2016/4/40/T--Groningen--layersguy.png" /> | + | <img src="https://static.igem.org/mediawiki/2016/a/a1/T--Groningen--Bacillus-couple.png" /> |
| </div> | | </div> |
| </div> | | </div> |
| | | |
− | <p>We followed two main ideas, namely hiding the key and deleting | + | <div class="split"> |
− | the key when unauthorized parties handle it.</p>
| + | <div class="right fltwo"> |
| + | <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><ul> |
| + | <li><a href="/Team:Groningen/BrickCharacter">Read more about how we determined the transformation efficiency..</a></ul></li> |
| + | </p></ul> |
| + | </div> |
| + | <div class="left flone"> |
| + | <img src="https://static.igem.org/mediawiki/2016/8/86/T--Groningen--TransformEffK8-4.jpg" /> |
| + | </div> |
| + | </div> |
| | | |
− | <h4>Decoy</h4> | + | <div class="split"> |
− |
| + | <div class="left fltwo"> |
− | <p>Hiding the key became called the <em>decoy</em> approach.The
| + | <h2>Key hiding</h2> |
− | 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
| + | <h4>Decoy</h4> |
− | recipient to activate the added ciprofloxacin and thus start
| + | |
− | selection of the right spore strain.</p>
| + | |
| | | |
− | <p>Our design of the decoy approach included the biobricks for
| + | <p>Hiding the key became called the <em>decoy</em> |
− | ciprofloxacin resistance, a super folder GFP and a pATP promotor.</p>
| + | 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> |
| | | |
− | <h4>Key deletion</h4> | + | <p>Only the knowledge about the right wavelength allows the |
| + | recipient to activate the added ciprofloxacin and thus |
| + | start selection of the right spore strain.</p> |
| + | |
| + | <p>Our design of the decoy approach included the biobricks |
| + | for ciprofloxacin resistance, a superfolder GFP and a pATP |
| + | promotor.</p> |
| + | |
| + | <p><ul> |
| + | <li><a href="/Team:Groningen/Decoy">Read more about the decoy experiment.</a></ul></li> |
| + | </u></p> |
| + | </div> |
| + | <div class="right flone"> |
| + | <img src="https://static.igem.org/mediawiki/2016/d/d3/T--Groningen--Decoy-4_2.jpg" /> |
| + | </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> |
| + | <li><a href="/Team:Groningen/PhotoswitchableAntibiotics">Read more about the MIC and MBC experiments..</a></ul></li> |
| + | </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> |
| | | |
− | <p>Another biological security layer is provided by our <em>key | + | <div class="split"> |
− | deletion system</em>. We have been working on two different approaches.
| + | <div class="right fltwo"> |
− | The first is a nucA killswitch which is made out of an assembly of
| + | <h2>Key deletion</h2> |
− | different biobricks. Atc or tetracycline have to be added to
| + | |
− | inhibit the tetR promoter to stop the expression of the nucA and
| + | <h4>NucA key deletion</h4> |
− | digestion of the key sequence.</p>
| + | |
| | | |
− | <p>The second approach makes use of a CRISPRcas system which will
| + | <p>Another biological security layer is provided by our key |
− | delete the key from the genome if no special treatment is applied.
| + | deletion system. This assures that only authorized parties |
− | Addition of Atc or tetracycline will stop the cas9 expression.</p>
| + | 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.</p> |
| | | |
− | <p>This system is highly flexible and biological layers can easily | + | <h4>CRISPR/Cas9 key deletion</h4> |
− | be added or modified to the wishes of the user.</p>
| + | |
| + | <p>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.</p> |
| + | |
| + | <p><ul> |
| + | <li><a href="/Team:Groningen/KeyDeletion">Read more about the key deletion.</a></ul></li> |
| + | </ul></p> |
| + | </div> |
| + | <div class="left flone"> |
| + | <img src="https://static.igem.org/mediawiki/2016/4/4f/T-Groningen-nucA-in-pSB1C3-plate2.jpg" /> |
| + | </div> |
| + | </div> |
| </section> | | </section> |
| </article> | | </article> |
| </html> | | </html> |
− | {{Groningen/Proof}}
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− | <!--{{Groningen/CiproResistance}}-->
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− | {{Groningen/Decoy}}
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− | {{Groningen/BrickCharacter}}
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| {{Groningen/footer}} | | {{Groningen/footer}} |