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| + | <!-- Spore movie: https://static.igem.org/mediawiki/2016/1/11/T--Groningen--Spore-Movie.mp4 --> |
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| <article> | | <article> |
| <section> | | <section> |
− | <h2>Experiments & Labjournal</h2> | + | <h2>Experiments</h2> |
| | | |
− | <p>Intro</p> | + | <p>The biological realization of CryptoGErM consisted of four |
− | </section>
| + | subprojects: integration, characterization, key hiding and key |
− | </html> | + | deletion. The encrypted message and the key were integrated into |
− | <!-- == Keys == -->
| + | the genome of <em>B. subtilis</em>. The location in the genomic DNA |
− | {{Groningen/Labjournal/Key-in-pDR111}}
| + | makes it harder to retrieve the data, since whole genome sequencing |
− | {{Groningen/Labjournal/Key-in-pSB1C3}}
| + | is required. The message is protected by a digital lock: the key, |
− | {{Groningen/Labjournal/Key-in-BBa_K823023}}
| + | and thus doesn’t need further biological protection. The key is not |
− | <!-- == Messages == -->
| + | encrypted and has to be protected by a biological lock. We followed |
− | {{Groningen/Labjournal/Message-in-pDR111}}
| + | different approaches to design a multi-layered biological lock. We |
− | {{Groningen/Labjournal/Message-in-pSB1C3}}
| + | followed two main ideas, namely hiding the key and deleting the key |
− | {{Groningen/Labjournal/Message-in-K823023}}
| + | when unauthorized parties handle it. This system is highly flexible |
− | <!-- == sfGFP == --> | + | and biological layers can easily be added or modified to the wishes |
− | <!-- in pDR111 -->
| + | 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> |
− | {{Groningen/Labjournal/sfGFP(Sp)-in-pSB1C3}}
| + | |
− | {{Groningen/Labjournal/sfGFP(Sp)-in-pDR111-pDR111-message-plasmid}}
| + | <div class="split"> |
− | <!-- == PatpI == --> | + | <div class="left flone"> |
− | {{Groningen/Labjournal/PatpI-in-pSB1C3}}
| + | <h2>Integration</h2> |
− | <!-- == Qnrs1 == --> | + | |
− | <!-- pSB1C3 --> | + | |
− | <!-- K8 --> | + | |
− | <!-- == nucA == --> | + | |
− | {{Groningen/Labjournal/nucA-in-pSB1C3}}
| + | |
− | {{Groningen/Labjournal/improved-nucA-in-pSB1C3}}
| + | |
| | | |
− | <html> | + | <p>The first subproject is the <em>integration</em>, the key and message |
− | <!--
| + | sequences were integrated into the genomic DNA of two separate |
− | <section class="collapse">
| + | <em>Bacillus subtilis</em> strains. In order to achieve this we had the key |
− | <h3>(title)</h3>
| + | and the message sequence synthesized by IDT. Then we cloned it in |
| + | 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 |
| + | integrate sequences into the amyE locus of <em>Bacillus subtilis</em>. From |
| + | there we integrated it in the genome of <em>B. subtilis</em> 168 trp+. For |
| + | message and key transmission these <em>B. subtilis</em> strains were |
| + | 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></li> |
| + | </ul></p> |
| + | </div> |
| + | <div class="right flone"> |
| + | <img src="https://static.igem.org/mediawiki/2016/a/a1/T--Groningen--Bacillus-couple.png" /> |
| + | </div> |
| + | </div> |
| | | |
− | <p>(intro description)</p> | + | <div class="split"> |
| + | <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> |
| | | |
− | <figure>
| + | <p><ul> |
− | <img src="(image url)" /> | + | <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> |
| + | |
| + | <div class="split"> |
| + | <div class="left fltwo"> |
| + | <h2>Key hiding</h2> |
| | | |
− | <figcaption>(Figure x caption)</figcaption>
| + | <h4>Decoy</h4> |
− | </figure>
| + | |
| | | |
− | <div class="collapse-h4">
| + | <p>Hiding the key became called the <em>decoy</em> |
− | <h4>(Experiment title)</h4>
| + | 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> |
| | | |
− | <h5>Experiment:</h5>
| + | <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>(text)</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> |
| | | |
− | <h5>PCR mixture:</h5> | + | <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> |
| + | |
| + | <div class="split"> |
| + | <div class="right fltwo"> |
| + | <h2>Key deletion</h2> |
| | | |
− | <p>(text) <a href="/Team:Groningen/Protocols#protocol-id">(link to protocol)</a></p>
| + | <h4>NucA key deletion</h4> |
− | | + | |
− | <h5>PCR set-up:</h5>
| + | <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. 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> |
| | | |
− | <h5>DNA Electrophoresis:</h5>
| + | <h4>CRISPR/Cas9 key deletion</h4> |
− | | + | |
− | <p>(text or something)</p>
| + | <p>The second approach makes use of a CRISPR/Cas9 system |
− | | + | which will delete the key from the genome if no special |
− | <h5>Conclusion:</h5>
| + | treatment is applied. Addition of aTc or tetracycline will |
− | | + | stop the Cas9 expression.</p> |
− | <p>(something or text)</p>
| + | |
− | | + | |
− | <h5>Procedure after gel validation:</h5>
| + | |
| | | |
− | <p>(washed hands)</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> | | </div> |
| </section> | | </section> |
− | -->
| |
| </article> | | </article> |
| </html> | | </html> |
| {{Groningen/footer}} | | {{Groningen/footer}} |