Difference between revisions of "Team:DTU-Denmark/Journal"

 
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                  >Home
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                  >Team
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Attributions"
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                  >Attributions
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                  </a></li></ul></li>
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                  aria-expanded="false">Project
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                 <li >
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                <a href="/Team:DTU-Denmark/Project/Overview"
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                  >Overview
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Project/Background"
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                  >Background
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Project/MAGE"
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                  >MAGE subtilis
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Project/Tyrocidine"
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                  >Tyrocidine
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                  </a></li>
 +
                <li >
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                <a href="/Team:DTU-Denmark/Project/Chip"
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                  >Lab-on-a-disc
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                  </a></li>
 +
                <li >
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                <a href="/Team:DTU-Denmark/Project/Inteins"
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                  >Inteins
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                  </a></li>
 +
                <li >
 +
                <a href="/Team:DTU-Denmark/Project/Detection"
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                  >Detection of NRP
 +
                  </a></li></ul></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Practices"
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                  >Human Practices
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                  </a></li>
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                  aria-expanded="false">Parts Collection
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                <li >
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                <a href="/Team:DTU-Denmark/Parts"
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                  >Parts
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Description"
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                  >Characterisation of xylR
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                  </a></li></ul></li>
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                <a href="/Team:DTU-Denmark/Journal"
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                  >Journal
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Software"
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                  >Software
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Achievements"
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                  >Key Achievements
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Collaborations"
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                  >Collaborations
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                  </a></li>
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                <li >
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                <a href="/Team:DTU-Denmark/Judging_Form"
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                  >Judging Form
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              <a href="/Team:DTU-Denmark/Team">Team</a>
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              <a href="/Team:DTU-Denmark/Journal">Journal</a>
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              <a href="/Team:DTU-Denmark/Part_Collection">Parts</a>
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              <a href="/Team:DTU-Denmark/Achievements">Achievements</a>
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          <li><a class="page-scroll" href="#Summary">Summary</a></li><li><a class="page-scroll" href="#Methods">Methods</a></li><li><a class="page-scroll" href="#Protocol">Protocol</a></li><li><a class="page-scroll" href="#Timeline">Timeline</a></li>
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      <li><a class="page-scroll" href="#Protocols">Protocols</a></li><li><a class="page-scroll" href="#LabNotebook">Lab Notebook </a></li><li><a class="page-scroll" href="#Timeline">Timeline</a></li>
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            <h1>Journal</h1>
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       <h1>
 
       <h1>
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         Summary
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         Protocols
 
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      <p>For OGRE (Oligo Recombineering) to be effective there is two genetic improvements that can be done. The first and most important is that the organism need to express a recombinase protein such as the beta protein derived from the lambda RED E. coli phage. The second improvement is to destroy/inhibit the mismatch repair system [-1]. In B. subtilis the genes mutS and mutL are taking care of the mismatch repair [1]. For proof of concept purposes deleting the mismatch repair system will be adequate. According to (REF) the protein encoded by mutL will not be functional without a functional mutS, this is why deletion of only mutS will be sufficient to take out the mismatch repair system. With respect to recombinase we will test two different recombinases: lambda-beta (codon optimized for <em>B. subtilis 168</em>) and gp35 (a recombinase from the B. subtilis phage SPP1) [-1]. For duing proof of concept of OGRE in <em>B. subtilis</em> four different strains was producere via genetic recombineering. All derived from <em>Bacillus subtilis 168</em>:</p>
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<h4 class="panel-title"><a aria-controls="collapseOne" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseOne" role="button">1% Agarose Gel</a></h4>
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<li>∆<em>amyE::beta-neo<sup>R</sup></em></li>
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<div class="panel-body">
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<li>∆<em>amyE::GP35-neo<sup>R</sup></em></li>
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<div class="panel-body">Needed materials: 100 ml&nbsp;TAE buffer,&nbsp;1gr. agarose,&nbsp;ethidium bromide.</div>
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<li>∆<em>mutS::beta-neo<sup>R</sup></em></li>
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<li>∆<em>mutS::GP35-neo<sup>R</sup></em></li>
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</ul>
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<p>The four different strains where compared to each other to get compare there transformation efficienc in MAGE.</p>
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<div class="panel-body">Mix 100 ml. TAE buffer with 1 gr. agarose powder, heat in microwave till the agarose is disolved, let it cool till it can be handled with bare hands, add 10 &micro;l&nbsp;ethidium bromide. Then&nbsp;pour the mixture&nbsp;into a mold containing slots for wells, and let cool.</div>
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<h4 class="panel-title"><a aria-controls="collapseTwo" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseTwo" role="button">Agarose Gel Electrophoresis</a></h4>
 
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      <p><strong>making beta and GP35</strong></p>
 
  
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<p>The beta CDS was codon optimized with JCat() and ordered from COMPANY. GP35 sequence was found from (GP35 article). The sequence was ordered from the same company as beta.</p>
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<div class="panel-body">
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<div class="panel-body">Set gel into electrophoresis chamber filled with 1X TAE buffer so the wells are covered.&nbsp;<br />
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Mix 5 &micro;l of&nbsp;your PCR reactions/sample with 1&nbsp;&micro;l 6X loading buffer.<br />
 +
Pipette 5&nbsp;&micro;l into each well,&nbsp;pour 2-3&nbsp;&micro;l ladder in some so the size can be seen.<br />
 +
Run at 140 V, for 30-40 min.</div>
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</div>
  
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<p><strong>incertion of recombinases in the amyE locus of B. subtilis</strong></p>
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<h4 class="panel-title"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseThree" role="button">Chemocompetent cells</a></h4>
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</div>
  
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<p>Beta and GP35 was inserted into the plasmid known as pDG268neo by using PCR with G5 HF 2x master mix from NEB (primer sequence hyperlink?). The plasmid was transformed into E coli DH5alpha by heat chock and incubated over night. The plasmid was purified from E coli by using QiAPrep mini prep kit. Digesting with restriction enzymes validated the insertion. This plasmid integrates the CDS into the amyE locus in Bacillus subtilis. pDG268neo with ether beta or GP35 was linearized and transformed into B subtilis W168 using the CFrench protocol (make CFrench to hyperlink)</p>
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<div class="panel-body">Chemo competens is a way of getting E. coli to be able to take up new DNA, since this cant be done naturally like B. subtilis, then chemocompetent cells have to be made.</div>
  
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<p>chromosomal DNA from B. subtilis 168 was isolated <strong>(protocol)</strong></p>
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<div class="panel-body">Protocol can be found&nbsp;<a href="https://static.igem.org/mediawiki/2015/c/cd/DTU-Denmark_Chemocompetentcells_protocol.pdf" target="_blank">here</a></div>
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</div>
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<p><strong>knock out mutS </strong></p>
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<h4 class="panel-title"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseFour" role="button">Glycerol stock</a></h4>
 +
</div>
  
−
<p>The regions upstream and downstream of the mutS locus were amplified by PCR from the chromosomal DNA with primers that had overlap with GP35 and beta.</p>
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<div aria-labelledby="headingFour" class="panel-collapse collapse" id="collapseFour" role="tabpanel">
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<div class="panel-body">
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<div class="panel-body">Needed materials: a 40% glycerol solution, bacteria of your choice, cryo tubes.</div>
  
−
<p>GP35 and beta were PCR amplified with primers that had overhangs that were homologies to the mutS locus in bacillus subtilis. GP35 and beta was assembled into the pSB1C3 plasmid by using gibson assembly and transformed into E coli and incubated over night. The plasmid was isolated by using mini prep.&nbsp; The plasmids were verified using restriction enzymes.</p>
+
<div class="panel-body">Mix glycerol and bacteria 50/50 in a cryo tube and place in a -80 frezzer till needed.</div>
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<p>beta and GP35 in pSB1C3 was linearized using XhoI, This also removed the Chloramphenicol gene from the plasmid. Beta and GP35 was transformed into B. subtilis.</p>
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</div>
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<p>&nbsp;</p>
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<p><strong>Genomic DNA extraction (B. brevis)</strong></p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p><strong>Ligation of DNA</strong></p>
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<p>&nbsp;</p>
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<p><strong>PCR</strong> -</p>
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<ul>
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<li>PCR</li>
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<li>Colony PCR</li>
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<li>Touch down</li>
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<li>&hellip;</li>
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<p><strong>Plasmid Purification Miniprep</strong></p>
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<p>&nbsp;</p>
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<p><strong>Restriction enzyme digest (NEB)</strong></p>
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<p><strong>Tyrocidine construct</strong></p>
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<p><strong>Transformation and Preparation of E.coli</strong><br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; -Preparation of Competent Cells<br />
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; -Transformation of Chemically Competent E.coli<br />
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<strong>Transformation of Bacillus</strong></p>
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+
−
<p><strong>Optiminsing OGRE in Bacillus</strong></p>
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−
<p>Different amount of oligo was tested to optimize OGRE method for Bacillus subtilis.</p>
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+
−
<p>The mismatch insertion frequency and oligo length was tested. The standard OGRE protocol was follow with the mismatches verified from 1-6 mismatches and oligoes from 50-100nt.</p>
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+
−
<p>OGRE with 4 cycles were attempted every cycle to approximately 6 hours. This was done to test if the transformation efficiency would rise when more cycles were attempted</p>
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<p>&nbsp;</p>
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<p><strong>changing surfatin</strong></p>
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+
−
<p>The 5th A domain in the NRPS for surfactin was attempted changed from a aspartate to a asparagine. This was done by following the OGRE protocol using a oligo that would change the specificity from asp to asn.</p>
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      <h1>
 
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        Protocol
 
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<h4 class="panel-title"><a aria-controls="collapseOne" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseOne" role="button">making plates</a></h4>
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<h4 class="panel-title"><a aria-controls="collapseFive" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseFive" role="button">Making plates</a></h4>
 
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<div class="panel-body">
 
<p>A lot of lab work requires plates in order to produce results, and often you need a special concentration, a unique antibiotic mixed into the plates or a different composition than what is available at the lab you are in. If that is the case, the plate needs to be made from scratch, or somewhat along the way.</p>
 
<p>A lot of lab work requires plates in order to produce results, and often you need a special concentration, a unique antibiotic mixed into the plates or a different composition than what is available at the lab you are in. If that is the case, the plate needs to be made from scratch, or somewhat along the way.</p>
  
−
<p>protocolcan be found <a href="https://static.igem.org/mediawiki/2015/b/b4/DTU-Denmark_Making_plates.pdf target="_blank">here</a></p>
+
<p>Protocol can be found <a href="https://static.igem.org/mediawiki/2015/e/e2/DTU-Denmark_making_plates_protocol.pdf" target="_blank">here</a>.</p>
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<h4 class="panel-title"><a aria-controls="collapseTwo" aria-expanded="false" class="collapsed" data-parent="#accordion2" data-toggle="collapse" href="#collapseTwo" role="button">Minimal meidia</a></h4>
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<h4 class="panel-title"><a aria-controls="collapseSix" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseSix" role="button">Minimal media</a></h4>
 
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<div class="panel-body">For our experiments we have used minimal media designed for B. subtilis. The plates have been made by following this protocol (for 1 liter)</div>
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<div class="panel-body">
 +
<div class="panel-body">For the&nbsp;experiments a minimal media designed for <em>B. subtilis</em> has been used. The plates have been made by following this protocol (for 1 liter).</div>
  
−
<div class="panel-body">protocolcan be found <a href="/static/Minimal_media.pdf target="_blank">here</a></div>
+
<div class="panel-body">Protocol can be found <a href="https://static.igem.org/mediawiki/2015/5/52/DTU-Denmark_Minimal_media_protocol.pdf" target="_blank">here</a>.</div>
 
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<h4 class="panel-title"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion3" data-toggle="collapse" href="#collapseThree" role="button">Collapsible Group Item #3</a></h4>
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<h4 class="panel-title"><a aria-controls="collapseSeven" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseSeven" role="button">Miniprep</a></h4>
 
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<div aria-labelledby="headingSeven" class="panel-collapse collapse" id="collapseSeven" role="tabpanel">
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<div class="panel-body">333</div>
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<div class="panel-body">In order to work with plasmids, then they needs to be purified&nbsp;and condensed, this is done bu minipreping the bacteria, and afterwards the concentration can be investigating using&nbsp;NanoDrop&trade;.</div>
 +
 
 +
<div class="panel-body">Protocol can be found <a href="https://static.igem.org/mediawiki/2015/4/48/DTU-Denmark_Miniprep_protocol.pdf" target="_blank">here</a>.</div>
 
</div>
 
</div>
 
</div>
 
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<div aria-multiselectable="true" class="panel-group" id="accordion4" role="tablist">
 
 
<div class="panel panel-default">
 
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<div class="panel-heading" id="headingFour" role="tab">
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<div class="panel-heading" id="headingEight" role="tab">
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<h4 class="panel-title"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion4" data-toggle="collapse" href="#collapseFour" role="button">Collapsible Group Item #4</a></h4>
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<h4 class="panel-title"><a aria-controls="collapseEigth" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseEigth" role="button">Natural competent Bacillus subtilis 168</a></h4>
 
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<div aria-labelledby="headingEigth" class="panel-collapse collapse" id="collapseEigth" role="tabpanel">
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<div class="panel-body">444</div>
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<div class="panel-body">
 +
<div class="panel-body">Bacillus can be made competent allowing an&nbsp;easy alteration of&nbsp;the genome and&nbsp;so wanted mutaions can be inserted, this is a unique way of investigateing new pathways.</div>
 +
 
 +
<div class="panel-body">Protocol can be found&nbsp;<a href="https://static.igem.org/mediawiki/2015/2/29/DTU-Denmark_natural_competent_subtilis_protocol.pdf" target="_blank">here</a>.</div>
 
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<h4 class="panel-title"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion5" data-toggle="collapse" href="#collapseThree" role="button">Collapsible Group Item #5</a></h4>
+
<h4 class="panel-title"><a aria-controls="collapseNine" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseNine" role="button">MAGE in Bacillus</a></h4>
 
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<div aria-labelledby="headingNine" class="panel-collapse collapse" id="collapseNine" role="tabpanel">
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<div class="panel-body">555</div>
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<div class="panel-body">
 +
<div class="panel-body">Electrotoration of&nbsp;bacteria is used in order to introduce mutations in the lagging strand of the strain, this technique&nbsp;are used in order to incorporate wanted mutations in a strain of&nbsp;Bacillus subtilis 168.</div>
 +
 
 +
<div class="panel-body">Protocol can be found&nbsp;<a href="https://static.igem.org/mediawiki/2015/0/0e/DTU-Denmark_MAGE_in_baccilus.pdf" target="_blank">here</a>.</div>
 
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<h4 class="panel-title"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion6" data-toggle="collapse" href="#collapseFour" role="button">Collapsible Group Item #6</a></h4>
+
<h4 class="panel-title"><a aria-controls="collapseTen" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseTen" role="button">Preperation of Electro Competent&nbsp;Bacillus subtilis 168</a></h4>
 
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<div class="panel-body">666</div>
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 +
<div class="panel-body">Electro competent cells are used in our MAGE part of the project, so a batch of cells was prepared using the following protocol.</div>
 +
 
 +
<div class="panel-body">Protocol can be found&nbsp;<a href="https://static.igem.org/mediawiki/2015/2/2e/DTU-Denmark_Electro_competent_protocol.pdf" target="_blank">here</a></div>
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<h4 class="panel-title"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion7" data-toggle="collapse" href="#collapseThree" role="button">Collapsible Group Item #7</a></h4>
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        Lab Notebook
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<h4 class="panel-title"><a aria-controls="collapseTwentyone" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseTwentyone" role="button">Expression of Tyrocidine</a></h4>
 
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<div class="panel-body">
 +
<div class="panel-body">The lab book for expression of tyrocidine&nbsp;can be found <a href="https://static.igem.org/mediawiki/2015/c/cf/DTU-Denmark_Tyrocidine_labnotebook.pdf" onclick="window.open(this.href, '', 'resizable=no,status=no,location=no,toolbar=no,menubar=no,fullscreen=no,scrollbars=no,dependent=no'); return false;">here</a>.&nbsp;</div>
 
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<h4 class="panel-title"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion8" data-toggle="collapse" href="#collapseFour" role="button">Collapsible Group Item #8</a></h4>
+
<h4 class="panel-title"><a aria-controls="collapseTwentytwo" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseTwentytwo" role="button">Generation&nbsp;of oligo-competent <em>B.subtilis</em> strains</a></h4>
 
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<div class="panel-body">888</div>
+
<div class="panel-body">
 +
<div class="panel-body">The lab book for the generation of oligo-competent strains can be found <a href="https://static.igem.org/mediawiki/2015/7/75/DTU-Denmark_straingeneration.pdf">here</a>.</div>
 
</div>
 
</div>
 
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<h4 class="panel-title"><a aria-controls="collapseThree" aria-expanded="false" class="collapsed" data-parent="#accordion9" data-toggle="collapse" href="#collapseThree" role="button">Collapsible Group Item #9</a></h4>
+
<h4 class="panel-title"><a aria-controls="collapseTwentythree" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseTwentythree" role="button">Blood cell lysis</a></h4>
 
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<div aria-labelledby="headingTwentythree" class="panel-collapse collapse" id="collapseTwentythree" role="tabpanel">
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<div class="panel-body">999</div>
+
<div class="panel-body">
 +
<div class="panel-body">To approve a lab-on-a-disc concept as a screening device, the UV-vis abasobancee were measured&nbsp;<a href="https://static.igem.org/mediawiki/2015/6/62/DTU-Denmark_bloodcell_lysis.pdf">here</a>.&nbsp;</div>
 
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<h4 class="panel-title"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion10" data-toggle="collapse" href="#collapseTen" role="button">Collapsible Group Item #10</a></h4>
+
<h4 class="panel-title"><a aria-controls="collapseTwentyfour" aria-expanded="false" class="collapsed" data-parent="#accordion" data-toggle="collapse" href="#collapseTwentyfour" role="button">MAGE&nbsp;proof of concept</a></h4>
 
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<div class="panel-body">000.</div>
+
<div class="panel-body">
 +
<div class="panel-body">See our labnotebook <a href="https://static.igem.org/mediawiki/2015/1/10/DTU-Denmark_labnote_MAGE_proof.pdf">here.</a></div>
 
</div>
 
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     <div class="row col-md-12">
 
     <div class="row col-md-12">
 
       <h1>
 
       <h1>
 
         Timeline
 
         Timeline
 
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       </h1>
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     </div>
 
     </div>
 +
   
 +
    <ul class="timeline">
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      <li>
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        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Biology of the Future </h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-09-14</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p style="text-align: justify;"><img alt="" src="https://static.igem.org/mediawiki/2015/c/c5/DTU-Denmark_timeline_novoamb.png" style="height: 158px; width: 250px; float: left;" />Our team member Pernille spent an afternoon giving a presentation on cell factories and synthetic biology to 30 highschool students. She says: &quot;Synthetic biology is all about being creative and use your imagination&nbsp;to design bio-solutions that can impact the world. For that exact reason, it is neccesary to discuss which ethical values tat should drive research&quot;. The highschool students had lots of questions and opinion for the discussion about ethics.</p>
 +
 +
<p style="text-align: justify;">&nbsp;</p>
 +
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          </div>
 +
        </div>
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      </li>
 +
   
 +
      <li class="timeline-inverted">
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        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">VWR to the rescue! </h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-09-01</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that the VWR&nbsp;is contributing our team with&nbsp;their awesome products!</p>
 +
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/3/37/DTU-Denmark_VWR_lab.jpg" style="width: 400px; height: 533px;" /></p>
 +
 +
<p>&nbsp;</p>
 +
 +
          </div>
 +
        </div>
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 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Visit from Fisher Scientific! </h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-26</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>Today we had a visit by&nbsp;Susanne Basse from&nbsp;Fisher Scientific. She&nbsp;came by our office with a wagon full of items, Thanks!&nbsp;</p>
 +
 +
<p><img alt="" src="/files/Fisher%20Scientific%20visit.png" style="width: 300px; height: 280px;" /></p>
 +
 +
          </div>
 +
        </div>
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      </li>
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      <li class="timeline-inverted">
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 +
        <div class="timeline-panel">
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contribution from Macrogen!</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-20</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are pleased&nbsp;to announce that Macrogen is sponsoring our team with sequencing!</p>
 +
 +
<p><a href="http://www.macrogen.com/" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/1/11/DTU-Denmark_Marcogen.jpg" style="width: 300px; height: 199px;" /></a></p>
 +
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          </div>
 +
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      <li>
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contributions from AKG!</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-19</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that AKG Acoustics is sponsoring our team with headphones for presentations in Boston!&nbsp;</p>
 +
 +
<p><a href="http://us.akg.com/akg-homepage-us.html" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/9/9d/DTU-Denmark_AKG.png" style="width: 400px; height: 121px;" /></a></p>
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      <li class="timeline-inverted">
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 +
        <div class="timeline-panel">
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contributions from VWR - Bie &amp; Berntsen A/S</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-11</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that VWR - Bie &amp; Berntsen A/S is sponsoring our team with lab consumeables!</p>
 +
 +
<p><a href="dk.vwr.com" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/e/ea/DTU-Denmark_VWR.jpg" style="width: 400px; height: 76px;" /></a></p>
 +
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          </div>
 +
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      <li>
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        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contributions from Fisher Scientific</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-11</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that Fisher Scientific&nbsp;is sponsoring our team with lab materials!&nbsp;</p>
 +
 +
<p><a href="https://dk.fishersci.com/dk/" target="_blank"><img alt="" src="/files/Fisher%20Scientific.jpg" style="width: 400px; height: 123px;" /></a></p>
 +
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      </li>
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      <li class="timeline-inverted">
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Canoeing</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-07</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>Today we took a day off from lab to go canoeing, followed by a barbeque at Chris&#39; place.</p>
 +
 +
<p><img alt="" src="https://static.igem.org/mediawiki/2015/6/6a/DTU-Denmark_Teambuilding.jpg" /></p>
 +
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          </div>
 +
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      </li>
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      <li>
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contribution from SnapGene</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-08-03</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that the SnapGene&nbsp;is contributing&nbsp;our team with lisences to their awesome product!</p>
 +
 +
<p><a href="http://www.snapgene.com/" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/5/58/DTU-Denmark_Snapgene.png" style="width: 400px; height: 113px;" /></a></p>
 +
 +
          </div>
 +
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      </li>
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 +
      <li class="timeline-inverted">
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 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Wiki Wizard</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-07-30</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We have uploaded the first beta version of the Wiki Wizard to<a href="https://github.com/dkmva/igem-wiki-wizard/commits/master"> <span class="fa fa-github " style="color:#000000;"></span>GitHub</a></p>
 +
 +
          </div>
 +
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      </li>
 +
   
 +
      <li>
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">First BioBrick cloned into B. subtilis</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-07-30</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>An expression cassette with lambda beta recombinase was sucesfully transformed into <em>Bacillus subtilis</em>. First step towards establishing MAGE&nbsp;in <em>Bacillus subtilis</em> completed!</p>
 +
 +
          </div>
 +
        </div>
 +
      </li>
 +
   
 +
      <li class="timeline-inverted">
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">BioBrick High School Project</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-07-23</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>3 high school students;&nbsp;Simran, Noor, and Charlotte, joined us and Biotech Academy for 5 days to make biosensors that changes color, when water is poluted. Read more at&nbsp;the <a href="/Team:DTU-Denmark/Practices" target="_blank">Human Practice</a>&nbsp;and <a href="http://biotechacademy.dk">http://biotechacademy.dk</a>.</p>
 +
 +
<p style="text-align: center;"><img alt="" src="https://static.igem.org/mediawiki/2015/b/b8/DTU-Denmark_highschool_biotechacademy.jpg" style="width: 350px; height: 197px;" /></p>
 +
 +
          </div>
 +
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      </li>
 +
   
 +
      <li>
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">First sequencing results</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-07-22</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We received our first sequencing results. One step closer to our&nbsp;first BioBrick!</p>
 +
 +
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 +
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      <li class="timeline-inverted">
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Brevibacillus parabrevis arrives</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-07-16</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p><em>Brevibacillus parabrevis </em>produces a lot of different antibiotics including tyrocidine. We will transfer the tyrocidine operon to our chassis <em>Bacillus subtilis</em>.</p>
 +
 +
          </div>
 +
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      </li>
 +
   
 +
      <li>
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contribution from the Lundbeck Foundation!</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-07-15</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that the Lundbeck Foundation is contributing&nbsp;financially to our team!</p>
 +
 +
<p><a href="http://www.lundbeckfoundation.com/" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/e/e2/DTU-Denmark_Lundbeckfonden.jpg" style="width: 348px; height: 96px;" /></a></p>
 +
 +
          </div>
 +
        </div>
 +
      </li>
 +
   
 +
      <li class="timeline-inverted">
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contributions from In Vitro!</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-05-06</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that In Vitro A/S is sponsoring our team with lab consumeables!&nbsp;</p>
 +
 +
<p><a href="in-vitro.dk" target="_top"><img alt="" src="/files/In%20Vitro.jpg" style="width: 400px; height: 156px;" /></a></p>
 +
 +
          </div>
 +
        </div>
 +
      </li>
 +
   
 +
      <li>
 +
        <div class="timeline-badge"><i class="glyphicon glyphicon-check"></i></div>
 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">Contributions from Frisnette</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-04-30</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that Frisnette is sponsoring our team with lab consumeables!&nbsp;</p>
 +
 +
<p><a href="http://frisenette.dk/" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/c/c8/DTU-Denmark_Frisenette.jpg" style="width: 330px; height: 85px;" /></a></p>
 +
 +
          </div>
 +
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      </li>
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      <li class="timeline-inverted">
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 +
        <div class="timeline-panel">
 +
          <div class="timeline-heading">
 +
            <h4 class="timeline-title">We get our strain</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-04-28</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <div class="pull-left"><img alt="" src="https://static.igem.org/mediawiki/2015/e/e1/DTU-Denmark_timeline_W168.jpg" style="width: 120px; height: 160px;" /></div>
 +
 +
<div class="pull-left">
 +
<p>We get a plate of our chassis strain <em>Bacillus subtilis</em> W168.<br />
 +
We can now begin the work in the laboratory.</p>
 +
</div>
 +
 +
          </div>
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          <div class="timeline-heading">
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            <h4 class="timeline-title">BioBrick Workshop</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-04-24</small></p>
 +
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 +
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            <p style="text-align: center;"><img alt="" src="https://static.igem.org/mediawiki/2015/f/fe/DTU-Denmark_timeline_biobrickworkshop.jpg" style="width: 350px; height: 198px;" /></p>
 +
 +
<p>Southern University of&nbsp;Denmark and University of Copenhagen joined us for a three-day BioBrick tutorial, where we spent three days in the laboratory cloning, learning about iGEM, wiki design, and getting to know each other through social activities.</p>
 +
 +
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            <h4 class="timeline-title">Contribution from the Otto Mønsted Fonden!</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-03-08</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>We are happy to announce that the Otto M&oslash;nsted Fonden&nbsp;is contributing&nbsp;financially to our team!</p>
 +
 +
<p><a href="http://www.ottomoensted.dk/" target="_blank"><img alt="" src="https://static.igem.org/mediawiki/2015/3/35/DTU-Denmark_Ottomoensted.png" style="width: 400px; height: 85px;" /></a></p>
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          <div class="timeline-heading">
 +
            <h4 class="timeline-title">First official team meeting</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2015-01-14</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>Today we had our first official team meeting. We talked about the project and got to know each other.</p>
 +
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 +
            <h4 class="timeline-title">Introductory meeting</h4>
 +
            <p><small class="text-muted"><i class="glyphicon glyphicon-time"></i> 2014-11-25</small></p>
 +
          </div>
 +
          <div class="timeline-body">
 +
            <p>This day, last years team held an introductory session about iGEM for interested people.</p>
 +
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Latest revision as of 15:12, 9 November 2015


Journal

Scroll down for more

Protocols

Needed materials: 100 ml TAE buffer, 1gr. agarose, ethidium bromide.
Mix 100 ml. TAE buffer with 1 gr. agarose powder, heat in microwave till the agarose is disolved, let it cool till it can be handled with bare hands, add 10 µl ethidium bromide. Then pour the mixture into a mold containing slots for wells, and let cool.
Set gel into electrophoresis chamber filled with 1X TAE buffer so the wells are covered. 
Mix 5 µl of your PCR reactions/sample with 1 µl 6X loading buffer.
Pipette 5 µl into each well, pour 2-3 µl ladder in some so the size can be seen.
Run at 140 V, for 30-40 min.
Chemo competens is a way of getting E. coli to be able to take up new DNA, since this cant be done naturally like B. subtilis, then chemocompetent cells have to be made.
Protocol can be found here
Needed materials: a 40% glycerol solution, bacteria of your choice, cryo tubes.
Mix glycerol and bacteria 50/50 in a cryo tube and place in a -80 frezzer till needed.

A lot of lab work requires plates in order to produce results, and often you need a special concentration, a unique antibiotic mixed into the plates or a different composition than what is available at the lab you are in. If that is the case, the plate needs to be made from scratch, or somewhat along the way.

Protocol can be found here.

For the experiments a minimal media designed for B. subtilis has been used. The plates have been made by following this protocol (for 1 liter).
Protocol can be found here.
In order to work with plasmids, then they needs to be purified and condensed, this is done bu minipreping the bacteria, and afterwards the concentration can be investigating using NanoDrop™.
Protocol can be found here.
Bacillus can be made competent allowing an easy alteration of the genome and so wanted mutaions can be inserted, this is a unique way of investigateing new pathways.
Protocol can be found here.
Electrotoration of bacteria is used in order to introduce mutations in the lagging strand of the strain, this technique are used in order to incorporate wanted mutations in a strain of Bacillus subtilis 168.
Protocol can be found here.
Electro competent cells are used in our MAGE part of the project, so a batch of cells was prepared using the following protocol.
Protocol can be found here

Lab Notebook

The lab book for expression of tyrocidine can be found here. 
The lab book for the generation of oligo-competent strains can be found here.
To approve a lab-on-a-disc concept as a screening device, the UV-vis abasobancee were measured here. 
See our labnotebook here.

Timeline

  • Biology of the Future

    2015-09-14

    Our team member Pernille spent an afternoon giving a presentation on cell factories and synthetic biology to 30 highschool students. She says: "Synthetic biology is all about being creative and use your imagination to design bio-solutions that can impact the world. For that exact reason, it is neccesary to discuss which ethical values tat should drive research". The highschool students had lots of questions and opinion for the discussion about ethics.

     

  • VWR to the rescue!

    2015-09-01

    We are happy to announce that the VWR is contributing our team with their awesome products!

     

  • Visit from Fisher Scientific!

    2015-08-26

    Today we had a visit by Susanne Basse from Fisher Scientific. She came by our office with a wagon full of items, Thanks! 

  • Contribution from Macrogen!

    2015-08-20

    We are pleased to announce that Macrogen is sponsoring our team with sequencing!

  • Contributions from AKG!

    2015-08-19

    We are happy to announce that AKG Acoustics is sponsoring our team with headphones for presentations in Boston! 

  • Contributions from VWR - Bie & Berntsen A/S

    2015-08-11

    We are happy to announce that VWR - Bie & Berntsen A/S is sponsoring our team with lab consumeables!

  • Contributions from Fisher Scientific

    2015-08-11

    We are happy to announce that Fisher Scientific is sponsoring our team with lab materials! 

  • Canoeing

    2015-08-07

    Today we took a day off from lab to go canoeing, followed by a barbeque at Chris' place.

  • Contribution from SnapGene

    2015-08-03

    We are happy to announce that the SnapGene is contributing our team with lisences to their awesome product!

  • Wiki Wizard

    2015-07-30

    We have uploaded the first beta version of the Wiki Wizard to GitHub

  • First BioBrick cloned into B. subtilis

    2015-07-30

    An expression cassette with lambda beta recombinase was sucesfully transformed into Bacillus subtilis. First step towards establishing MAGE in Bacillus subtilis completed!

  • BioBrick High School Project

    2015-07-23

    3 high school students; Simran, Noor, and Charlotte, joined us and Biotech Academy for 5 days to make biosensors that changes color, when water is poluted. Read more at the Human Practice and http://biotechacademy.dk.

  • First sequencing results

    2015-07-22

    We received our first sequencing results. One step closer to our first BioBrick!

  • Brevibacillus parabrevis arrives

    2015-07-16

    Brevibacillus parabrevis produces a lot of different antibiotics including tyrocidine. We will transfer the tyrocidine operon to our chassis Bacillus subtilis.

  • Contribution from the Lundbeck Foundation!

    2015-07-15

    We are happy to announce that the Lundbeck Foundation is contributing financially to our team!

  • Contributions from In Vitro!

    2015-05-06

    We are happy to announce that In Vitro A/S is sponsoring our team with lab consumeables! 

  • Contributions from Frisnette

    2015-04-30

    We are happy to announce that Frisnette is sponsoring our team with lab consumeables! 

  • We get our strain

    2015-04-28

    We get a plate of our chassis strain Bacillus subtilis W168.
    We can now begin the work in the laboratory.

  • BioBrick Workshop

    2015-04-24

    Southern University of Denmark and University of Copenhagen joined us for a three-day BioBrick tutorial, where we spent three days in the laboratory cloning, learning about iGEM, wiki design, and getting to know each other through social activities.

  • Contribution from the Otto Mønsted Fonden!

    2015-03-08

    We are happy to announce that the Otto Mønsted Fonden is contributing financially to our team!

  • First official team meeting

    2015-01-14

    Today we had our first official team meeting. We talked about the project and got to know each other.

  • Introductory meeting

    2014-11-25

    This day, last years team held an introductory session about iGEM for interested people.

Technical University of Denmark
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark
P: +45 45 25 25 25
M: dtu-igem-2015@googlegroups.com