Difference between revisions of "Team:Wageningen UR/Notebook/Outreach"

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<h4><a href="#0">Cas9-based <br>kill switch</a></h4>
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<h4><a href="https://2016.igem.org/Team:Wageningen_UR/HP/Gold">Main Page</a></h4>
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<a href="#1">May 15 - May 25</a>
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<a href="#march">March</a>
 
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<a href="#2">May 26 - June 6</a>
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<a href="#april">April</a>
 
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<a href="#3">June 7 - June 29</a>
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<a href="#may">May</a>
 
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<a href="#4">June 30 - July 31</a>
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<a href="#june">June</a>
 
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<a href="#5">Aug 1 - Aug 31</a>
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<a href="#july">July</a>
 
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<a href="#6">Sept 1 - Okt 10</a>  
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<a href="#august">August</a>
 
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<a href="#7">References</a>  
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<a href="#september">September</a>
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<a href="#october">October</a> 
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<section id="march">
<p>Unless indicated otherwise, all experiments were performed by Belwina</p>
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<h2><b>March 23rd – Multi-Stakeholder Dialogue</b></h2>
 
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<h3><b>The Role of Synthetic Biology in Energy Transition </b></h3>
<section id="1">
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<p> To broaden our horizon and understanding of the possibilities of synthetic biology, we joined a multi-stakeholder dialogue that was organized to discuss the role of synthetic biology in energy transition, by the Athena institute and the Rathenau institute. There is a general belief that something needs to change about the current Dutch energy supply, and during this dialogue we brainstormed about how synthetic biology could be of help achieving this. In a guided way, we generated ideas about applications of synthetic biology for energy supply, starting from future society-scenario’s created the day before. The nice thing about this approach was that we learned about the perspectives of people with different backgrounds, ranging from scientists to business people. It felt like we all started on the same page. The ideas that were most appealing were later worked out in different groups. Lisa and I worked on a scenario, “the green human”, where humans would evolve together with plants to be able to rely on photosynthesis for energy. Of course this scenario is very futuristic, but in our opinion it exposes a very important aspect of both energy transition and synthetic biology: that the public should be involved, to be more conscious about energy use and more engaged with technological advances in the field of synthetic biology. </p>
<h1><b>May 15 - may 25</b></h1>
+
<section id="april">
<p>
+
<h2><b>April – Meetings with DAE</b></h2>
 
+
<p> As mentioned in the collaboration section, we designed our project with the help of students from the Design Academy Eindhoven. Apart from collaborating with them on designing the future end product BeeT, they helped us think about our project and its connection with society. We met with them twice: The first time, we visited them and took part in a design workshop. We talked about our ideas for our iGEM projects - at that time there were still a lot of ideas on the table. The students helped us realize what other “non-science” people think about synthetic biology. The second meeting was a lab day for them to see what our everyday life in the lab looks like. It was fun sharing our knowledge with them. On this day, we especially realized what kind of prejudices a lot of people have towards GMOs. At the end of the day, we learned what aspects we should pay special attention to when presenting our project to the broad public. Moreover, we were able to convince the students that synthetic biology is in fact something good rather than something threatening.
<b>Moving iGEM Cas9 to another backbone</b>
+
<br>
+
Belwina, Marijn and Thomas transformed biobrick parts and backbones from the registry to home-made chemically competent DH5alpha cells. Transformations were plated on LB agar plates with antibiotics corresponding to the resistance in the backbone of each plasmid.  
+
<br>
+
<br>
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Transformed parts:</p>
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+
<figure>
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<img src="https://static.igem.org/mediawiki/2016/2/26/T--Wageningen_UR--Belnote_biobricks.JPG">
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</figure><br/>
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+
<p>
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<br>
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The plasmids were <a href="https://static.igem.org/mediawiki/2016/b/bd/T--Wageningen_UR--MiniPrep.pdf">isolated</a> from liquid cultures inoculated from colonies of transformation plates. Also, glycerol stocks were made.  
+
The length of the dCas9 part (5080 bp) was verified using PCR with the VF2 and VR primer.  
+
 
</p>
 
</p>
  
<figure>
 
<img src="https://static.igem.org/mediawiki/2016/d/db/T--Wageningen_UR--Belnote_biobricksconfirm1.JPG">
 
</figure><br/>
 
 
<p><br>
 
<b>Moving dCas9 to pET26B</b>
 
<br>
 
E. coli glycerol stocks containing pdCas9 (Addgene plasmid # 46569) and pET26B were streaked on LB plates with the appropriate antibiotic, liquid cultures were inoculated and the plasmids were <a href="https://static.igem.org/mediawiki/2016/b/bd/T--Wageningen_UR--MiniPrep.pdf">isolated</a>.
 
<br>
 
<br>
 
Q5 PCR was performed on these plasmids, to make fragments with cleaving sides for PacI and SpeI:<br>
 
pdCas9:<br>
 
Fwd primer: Bel-cas9 Cas9 fwd (acacacTTAATTAATGACAGCTTATCATCGATAAGCT)<br>
 
Rev primer: Bel-cas9 Cas9 rev (acacacACTAGTTCAGTCACCTCCTAGCTGAC)<br>
 
Annealing T: 60ºC, elongation time 1.5 minutes.<br>
 
Expected band size: 5340 bp
 
<br>
 
<br>
 
pET26B:<br>
 
Fwd primer: Bel-cas9 pET fwd (acacacactagtgcgcaacgcaattaatgtaag)<br>
 
Rev primer: Bel-cas9 pET rev (acacacttaattaaatggatatcggaattaattcggatc)<br>
 
Annealing T: 60ºC, elongation time 1.5 minutes. <br>
 
Expected band size: 3737 bp
 
<br>
 
<br>
 
Fragments were checked on 1% agarose TAE gels, 100V, 30 minutes. The PCR for pET26B worked, whereas the one for pdCas9 did not. </p>
 
  
 
<figure>
 
<figure>
<img src="https://static.igem.org/mediawiki/2016/d/d7/T--Wageningen_UR--Belnote_caspetpcr1.JPG">
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<video width="720" controls="" style="display:block;margin-left:auto;margin-right:auto;">
</figure><br/>
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  <source src="https://static.igem.org/mediawiki/2016/c/c5/T--Wageningen_UR--DAEmovie.mp4" type="video/mp4">
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  <p>Your browser does not support this video. Please upgrade your browser.</p>
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</video>
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<figcaption>The movie Leif Czakai (DAE student) made from us, making baklava.</figcaption>
 +
</figure>
 +
<!-- no, maybe this can go on the main HO page. frame it like: this is our way to include artists in our work. Leif made this movie of us, working together on a project.  -->
  
<p>
 
<br>
 
<b>Collection and construction of pEVOL plasmids</b><br>
 
The following plasmids were received from Peter Schultz:<br>
 
pEVOL-pAzF (Addgene plasmid # 31186)<br>
 
pEVOL-pBpF (Addgene plasmid # 31190)<br>
 
As well as the recoded strain C321ΔA-exp, from George Church (Bacterial strain #49018)
 
All were delivered as agar stabs, that were streaked on LB plates with the appropriate antibiotic.
 
</section>
 
  
<section id="2">
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<section id="may">
<h1><b>May 26 - June 6</b></h1>
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<h2><b>May 21st – National Meetup</b></h2>
<p>
+
<p> On 21/05/2016 we organised the meet-up of all interested Dutch iGEM teams. Four teams took part: Groningen, Leiden, Eindhoven, and us. The meeting was divided in two parts. The first part consisted of every team presenting their project ideas with a brief discussion afterwards. After lunch the brainstorm session started. We divided the group in 4 random subgroups. The groups got to know eachother a bit, and then brainstormed on whatever iGEM related topic they liked. Some groups decided to talk about what team could collaborate with what other team, some teams decided to think about ways to connect in a better way, and some teams thought about how to make iGEM more popular in the Netherlands. After some small presentations of the brainstorm sessions’ results, we discussed several ideas. The outcome of the discussions was: <br>
<b>General</b><br>
+
• a list with contact details and team roles of every Dutch team member has been obtained <br>
Heat-shock competent cells were made according to the <a href="https://static.igem.org/mediawiki/2016/9/9c/T--Wageningen_UR--Preparing_and_transforming_chemically_competent_cells_using_the_BacGen_protocol.pdf">BacGen protocol</a>. After testing by transforming with PUC19, transformation efficiency turned out to be 10^4.  
+
• the notion of several collaboration possibilities <br>
<br>
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• ideas about shared sponsoring and funding <br>
<br>
+
When the discussions were over, the official part came to an end, but some teams ended up ordering pizza and playing board games together. All in all, we feel the day was a success not only for the progress of our projects, but also for getting to know and connecting with our fellow Dutch iGEM teams.  
<b>Moving iGEM Cas9 to another backbone</b><br>
+
</p>
iGEM Cas9, pSB1K3, pSB6A1 and pSB4K5 were <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digested</a> with EcoRI-HF and PstI-HF. Fragments were checked on agarose gels. Estimated band size:<br>
+
iGEM Cas9, 5080 bp<br>
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pSB1K3, 2204 bp<br>
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pSB6A1, 4022 bp<br>
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pSB4K5, 3419 bp<br>
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pSB1K3 certainly did not have the correct length, and the other two backbones were unclear, as they should have a different size but they did not. The digested iGEM Cas9 looked fine. </p>
+
  
<figure>
+
<section id="june">
<img src="https://static.igem.org/mediawiki/2016/4/4f/T--Wageningen_UR--Belnote_casandBB_dig.JPG">
+
<h2><b>June 24th & 25th – Synenergene Forum</b></h2>
</figure><br/>
+
<p>In search for the right conditions for responsible research and innovation, this conference was organized by the Rathenau institute (hyperlink) for all relevant stakeholders when it comes to the future of synthetic biology. Scientists, business people, policymakers and civil society organizations attended, but it was also open for the general public. The promises and pitfalls of synthetic biology were considered from a societal perspective during pitches, presentations, workshops and debates. We attended a workshop about connecting synthetic biology with culture and religion, a link we had not thought about before. During small information rounds, we had the opportunity to talk to experts in the field. We also joined a plenary discussion about freedom and security in an age of synthetic biology. We enjoyed hearing so many different opinions and having the opportunity to talk to experts from all over the world.</p>
  
<p>
 
<br>
 
iGEM Cas9, pSB1K3 and pSB4K5 were cut from the gel and purified (Machery-Nagel nucleospin kit). Samples were kept in the freezer.
 
<br>
 
<br>
 
After a few days, I tried <a href="https://static.igem.org/mediawiki/2016/7/76/T--Wageningen_UR--Ligation.pdf">ligating</a> iGEM Cas9 in pSB6A1 and pSB4K5 according to the standard protocol, transformed 2 µL into heat-shock competent cells (the ones mentioned above).
 
<br>
 
<br>
 
The ligation of Cas9 in pSB4K5 gave 7 colonies, but <a href="https://static.igem.org/mediawiki/2016/f/f8/T--Wageningen_UR--Colony_PCR.pdf">Colony PCR</a> (primers: VF2 and VR) revealed there was no insert (expected band: 5080). </p>
 
  
<figure>
+
<section id="july">
<img src="https://static.igem.org/mediawiki/2016/8/83/T--Wageningen_UR--Belnote_iCas9colonyPCR.JPG">
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<section id="august">
</figure><br/>
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<section id="september">
 
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<h2><b>September 20th – RIVM meeting</b></h2>
<p>
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<h3><b>Making the Safe by design movie </b></h3>
<br>
+
<p> In contrast to many other synthetic biology applications, BeeT is intended to be used outside the lab, in beehives, which is in close contact with nature. To get a better idea of the viability of our idea, and what it is like to request permission to use an engineered microorganism outside of the lab, we looked at the questions that are asked in such a request (link to website of RIVM) and discuss some that peaked our interest in the movie. Some interesting questions we had not thought about, such as “how can a genetically modified microorganism be recognized among natural relatives?”. Another interesting aspect of the application of synthetic biology is responsibility: if our engineered bacterium would be commercialized, who would be responsible for any adverse effects? In our opinion, scientists are responsible for thoroughly and objectively investigating and reporting possible risks. Companies in turn should monitor whether the technology is sufficiently researched. Companies should also make sure that user manuals are correct and clear to understand, even to people with little experience with comparable technology. Users of the product, the beekeepers in our case, are responsible for using the product in the intended way only and following the user manual provided with the product.  
Another approach was was PCR amplifying Cas9 and backbones with VF2 and VR primers. Only the pSB1K3 backbone and iGEM Cas9 (5380 bp) gave a convincing band, so these PCR products were purified (hyperlink), <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digested</a> with EcoRI-HF and PstI-HF, <a href="https://static.igem.org/mediawiki/2016/7/76/T--Wageningen_UR--Ligation.pdf">ligated</a> and <a href="https://static.igem.org/mediawiki/2016/0/00/T--Wageningen_UR--Preparing_and_transforming_DH5%CE%B1_electrocompetent_cells.pdf">transformed</a> into electrocompetent cells (made by Linea). 
+
Our movie ends with some open questions regarding safe by design, both in general and within our project. Examples are “what should be done to actually realize the insights that are gained by safe by design?” and “should there be more focus on the positive aspects of using a genetically modified microorganism when judging a permission request?”. We don’t know the answer to these questions, but think they are important nevertheless. </p>
<br>
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<br>
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<b>Moving dCas9 to pET26B<b><br>
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pdCas9 was verified using <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digestion</a> with SacI-HF & SalI-HF, as well as digestion with SacI-HF and XbaI.  
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<br>
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<br>
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The PCR for pdCas9 was repeated under various conditions; a range of annealing temperatures, addition of 5% DMSO, and varying template concentrations. Nothing worked. So I looked closer at the primers, and they turned out to be suboptimal in terms of stability of the 3’ vs 5’ end.<br>
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My supervisor helped me design better primers:<br>
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BelCas9-cas9Fw2 (GCCTTAATTAATGACAGCTTATCATCGATAAGCTTTAATG)<br>
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BelCas9-cas9Rev2 (GCCACTAGTAATTGCATCAACGCATATAGCGCTAGCAG)<br>
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<br>
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<br>
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Q5 PCR was performed on pdCas9 using these primers:<br>
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Annealing T: gradient ranging from 60-72ºC, elongation time: 1.5 minutes<br>
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Expected band size: 5349 bp
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<br>
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<br>
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And this time it worked. </p>  
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<figure>
 
<figure>
<img src="https://static.igem.org/mediawiki/2016/6/6c/T--Wageningen_UR--Belnote_dCas9_gradPCR.JPG">
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<video width="720" controls="" style="display:block;margin-left:auto;margin-right:auto;">
</figure><br/>
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  <source src="https://static.igem.org/mediawiki/2016/8/8b/T--Wageningen_UR--RIVMmovie.mp4" type="video/mp4">
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  <p>Your browser does not support this video. Please upgrade your browser.</p>
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</video>
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<figcaption>The movie we made in collaboration with the RIVM.</figcaption>
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</figure>  
  
<p>
 
<br>
 
PCR products of pdCas9 and pET26B were purified (hyperlink), <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digested</a> with PacI and SpeI, <a href="https://static.igem.org/mediawiki/2016/7/76/T--Wageningen_UR--Ligation.pdf">ligated</a> and <a href="https://static.igem.org/mediawiki/2016/0/00/T--Wageningen_UR--Preparing_and_transforming_DH5%CE%B1_electrocompetent_cells.pdf">transformed</a> into electrocompetent cells (made by Linea).
 
<br>
 
<br>
 
<b>Result of transformation of dCas9 and iGEM Cas9 ligations</b><br>
 
Basically all transformations had colonies, also control transformations with only backbone. Only transformations without ligase were clean, indicating there was probably some back-ligation of the backbones. 
 
<br>
 
<br>
 
<a href="https://static.igem.org/mediawiki/2016/f/f8/T--Wageningen_UR--Colony_PCR.pdf">Colony PCR</a>s with VF2, VR, BelCas9-cas9Fw2 and BelCas9-cas9Rev2 (for iGEM ligations and own ligations, respectively) revealed there were no clones with the correct insert. </p>
 
  
<p><br>
+
<h3><b>Meeting</b></h3>
I decided to focus on my own Cas9 construct from now on, and drop the iGEM construct.
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<p> Concluding our collaboration with the RIVM, some members of our team, as well as the other Dutch iGEM teams attended a meeting organized by the RIVM and the Rathenau institute, with the theme: “veilig verder met synthetische biologie”, or “making safe progress in synthetic biology”. During this meeting, researchers, policymakers and policy advisors brainstormed, discussed and presented their ideas about how synthetic biology should be handled in the future. We gave a short presentation about the topic of our project and how we integrated “safe by design” through toxin specificity, controlled expression of the toxin and confinement of our product to the beehive. During a short information market, we got to talk more in depth with people who were interested, presented the movie we made for the RIVM and the Resource from the future that was made in collaboration with Synenergene. There were short “break-out sessions” where safety of previous iGEM projects was discussed in small groups, and at the same time two artists made a huge and beautiful drawing of everything that was discussed during the day. We learned a lot from the interesting talks about further handling safety in synthetic biology both from a political and a societal point of view. An interesting point that comes to mind was made by Sabine Roeser (professor of Ethics, TU Delft), about how feelings of the public concerning synthetic biology can be a useful source of questions on the ethical aspects of our work. She also mentioned how art can be used to connect people to synthetic biology. In our project, we tried to achieve this through collaborating with the Design Academy Eindhoven (hyperlink). Besides, it was nice to get to know more about how the other Dutch iGEM teams handled the RIVM assignment. We thank the RIVM and the Rathenau institute for inviting us, it was an inspiring day! </p>
<br>
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<br>
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<b>pT7-gRNA construction</b><br>
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A glycerol stock of E. coli containing pT7-gRNA was streaked on LB agar with ampicillin and allowed to grow overnight. A colony was picked for making a glycerol stock, as well as plasmid isolation (Machery-Nagel nucleospin kit). </p>
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</section>
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<section id="3">
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<section id="october">
<h1><b>June 7 - June 29</b></h1>
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<p>No labwork due to moving of the lab.</p>
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+
</section>
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<section id="4">
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<h1><b>June 30 - July 31</b></h1>
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<p>
+
<b>General</b><br>
+
Electrocompetent E. coli cells were made according to the <a href="https://static.igem.org/mediawiki/2016/0/00/T--Wageningen_UR--Preparing_and_transforming_DH5%CE%B1_electrocompetent_cells.pdf">protocol</a>. Transformation efficiency was very high (could not count the colonies, really).
+
<br>
+
<br>
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<b>Moving dCas9 to pET26B</b><br>
+
New PCR products were made of pdCas9 and pET26B. I proceeded with purification and <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digestion</a> as usual, but added a step with alkaline phosphatase (CIP, NEB).
+
<br>
+
<br>
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Performed a href="https://static.igem.org/mediawiki/2016/7/76/T--Wageningen_UR--Ligation.pdf">ligation</a> and <a href="https://static.igem.org/mediawiki/2016/0/00/T--Wageningen_UR--Preparing_and_transforming_DH5%CE%B1_electrocompetent_cells.pdf">electroporation</a> of ligation. This time cloning was more successful: some colonies on ligation mixture, no colonies on backbone only control.
+
<br>
+
<br>
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<a href="https://static.igem.org/mediawiki/2016/f/f8/T--Wageningen_UR--Colony_PCR.pdf">Colony PCR</a> revealed that some colonies probably contained the correct construct. This was verified by sequencing. </p>
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+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/9/94/T--Wageningen_UR--Belnote_caspet_colPCR.JPG">
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</figure><br/>
+
 
+
<p>
+
<br>
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<b>Collection and construction of pEVOL plasmids</b><br>
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We received the biocontainment strains from Harvard, from which pEVOL-BipA was extracted (Machery-Nagel nucleospin kit).
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<br>
+
<br>
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<b>Construction of pT7-gRNA plasmids</b>
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The following primers were <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas3">annealed</a> as inserts:
+
<br>
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<br>
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pBbS5a (RFP) 1766-1785 FWD fwd
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5’- TAGGgtggtccgctgccgttcgct-3’
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<br>
+
<br>
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pBbS5a (RFP) 1766-1785 FWD rev
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5’- AAACagcgaacggcagcggaccac-3’
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<br>
+
<br>
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pBbS5a (RFP) 1739-1758 REV fwd
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5’- TAGGaactttcagtttagcggtct -3’
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<br>
+
<br>
+
pBbS5a (RFP) 1739-1758 REV rev
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5’- AAACagaccgctaaactgaaagtt -3’
+
<br>
+
<br>
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pBbS5a (RFP) 1821-1840 FWD fwd
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5’- TAGGcaaagcttacgttaaacacc -3’
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<br>
+
<br>
+
pBbS5a (RFP) 1821-1840 FWD rev
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5’- AAACggtgtttaacgtaagctttg -3’
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<br>
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<br>
+
pBbS5a (RFP) 1803-1822 REV fwd
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5’- TAGGtggaaccgtactggaactgc -3’
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<br>
+
<br>
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pBbS5a (RFP) 1803-1822 REV rev
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5’- AAACgcagttccagtacggttcca -3’
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<br>
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<br>
+
We tried at first constructing the pT7-gRNA plasmids using protocol described in Jao et al. (2014), but this gave a lot of false positive colonies (on plates transformed without any insert). <br>
+
Still, some plasmids were <a href="https://static.igem.org/mediawiki/2016/b/bd/T--Wageningen_UR--MiniPrep.pdf">isolated</a> from colonies of plates with insert, and <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digested</a> with SalI-HF and ScaI-HF. Any positive clones should not be cut by SalI, because this restriction site is only present in the original backbone. Expected bands: 759 bp and 1782 bp. No positive clones were found. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/6/64/T--Wageningen_UR--Belnote_gRNAfail.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
The next strategy was to <a href="https://static.igem.org/mediawiki/2016/a/ac/T--Wageningen_UR--Restriction_Enzyme_Digestion.pdf">digest</a> with BsmBI and SalI, <a href="https://static.igem.org/mediawiki/2016/b/b9/T--Wageningen_UR--Gel_Extraction_of_DNA.pdf">isolate the linearized plasmid from gel</a>, and proceed with a href="https://static.igem.org/mediawiki/2016/7/76/T--Wageningen_UR--Ligation.pdf">ligation</a>. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/0/0e/T--Wageningen_UR--Belnote_gRNA_dig.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
This did give us some positive clones. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/7/73/T--Wageningen_UR--Belnote_gRNA_success.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
Procedure was repeated for gRNA 3 and 4. Results were later confirmed by sequencing (however, it turned out that gRNA 3 was not correct after all. It took another round of picking colonies/digestion/sequencing before we also got that one right).
+
<br>
+
<br>
+
<b>Mutagenesis of dCas9-pET26B</b><br>
+
The Ala10TAG and Ala840TAG mutations were introduced by <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas1">mutagenesis PCR</a>, using the following primers:
+
<br>
+
<br>
+
dCas9 Ala10TAG fwd
+
5'-ggcaaaaatggataagaaatactcaataggcttatagatcggcacaaatagcgtc-3'
+
<br>
+
<br>
+
dCas9 Ala10TAG rev
+
5'-gacgctatttgtgccgatctataagcctattgagtatttcttatccatttttgcc-3'
+
<br>
+
<br>
+
dCas9 Ala840TAG fwd
+
5'-taatcgtttaagtgattatgatgtcgattagattgttccacaaagtttccttaaagacg-3'
+
<br>
+
<br>
+
dCas9 Ala840TAG rev
+
5'-cgtctttaaggaaactttgtggaacaatctaatcgacatcataatcacttaaacgatta-3'
+
<br>
+
<br>
+
First PCRs revealed that only the Ala840TAG PCR was successful, as was revealed by <a href="https://static.igem.org/mediawiki/2016/0/0a/T--Wageningen_UR--Gel_Electrophoresis.pdf">gel electrophoresis</a> (expected band size for both: 9077 bp). </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/0/09/T--Wageningen_UR--Belnote_mutpcr_1.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
The Ala10TAG mutation worked after addition of GC enhancer to the PCR mixture. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/a/a9/T--Wageningen_UR--Belnote_mutpcr_2.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
Mutations were verified after sequencing of <a href="https://static.igem.org/mediawiki/2016/b/bd/T--Wageningen_UR--MiniPrep.pdf">isolated</a> plasmids. </p>
+
</section>
+
 
+
<section id="5">
+
<h1><b>Aug 1 - Aug 31</b></h1>
+
 
+
<p>
+
<b>Expression of Cas9-pET26B in C321ΔA</b><br>
+
Cas9-pET26B constructs as well as iGEM-Cas9 and the original pdCas9 were transformed in E. coli C321ΔA as described in Lajoie <i>et. al</i> (2013) protocol<sup><a href="#bp1" id="refbp1">1</a></sup> for electroporation, successfully.
+
Later, also, pEVOL-BipA, pEVOL-pAzF and pEVOL-pBpF were transformed into C321ΔA, both with and without Cas9-pET26B constructs.
+
<br>
+
<br>
+
A first expression experiment was done with 50 ml overnight cultures of C321ΔA + Cas9 construct, in LB with the appropriate antibiotic.
+
<br>
+
Cells were spun down, resuspended in 10 ml lysis buffer (50mM Tris-HCL, 250 mM NaCl, 1mM EDTA) and lysed by sonication (4x15 sec, 25Am).
+
<br>
+
Protein concentrations were measured with a Bradford assay.</p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/a/ae/T--Wageningen_UR--Belnote_bradford_1.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
20 ug of each extract was loaded on SDS to check for Cas9 expression. The expected weight of Cas9 is 156 kDa, of dCas9-Ala10TAG is 1 kDa (can’t be seen anyways), and of dCas9-Ala840TAG it is 97 kDa. No such bands could be observed (possible also due to a background band of the same size)</p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/f/f0/T--Wageningen_UR--Belnote_sds_caspet.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
because for the original iGEM construct, the band seemed to be a bit more pronounced, I grew new cultures and repeated the experiment. This time, it was really obvious that expression levels were too low. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/6/67/T--Wageningen_UR--iGEMCas9SDS.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
<i><b>in vitro</i> transcription of guide RNAs</b>
+
By the time of transcription, guide 3 had not been verified by sequencing yet, so only guide 1, 2 and 4 were transcribed and purified, according to the <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas4">protocol</a>.
+
<br>
+
<br>
+
I only have a picture of the gel after cutting the RNA bands, but they were present. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/b/b0/T--Wageningen_UR--Belnote_gRNA_transcribed.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
After purification, guide 2 and 4 had decent concentrations of ~350 ng/uL. Guide 1 had only 35 ng/uL.
+
<br>
+
<br>
+
<b>Cloning of Cas9 variants in expresso vector</b><br>
+
After discussion with an employee in the departement who has experience expressing <i>Streptococcus pyogenes</i> Cas9, it was decided that expression from pdCas9 is probably too low to visualize on SDS-PAGE, and perhaps not suitable for further <i>in vitro</i> testing.
+
<br>
+
So it was decided to clone Cas9 in the <a href="http://www.lucigen.com/Expresso-Rhamnose-Cloning-and-Protein-Expression-System/">Expresso c-rham vector system</a>.
+
<br>
+
<br>
+
First, Cas9 variants and the Expresso vector were amplified by <a href="https://static.igem.org/mediawiki/2016/2/27/T--Wageningen_UR--Polymerase_Chain_Reaction.pdf">PCR</a>. The following reactions were performed:
+
<br>
+
<br>
+
Expresso<br>
+
fwd: CATCATCACCACCATCACTAATAG<br>
+
Rev: CATATGTATATCTCCTTCTTATAGTTAAAC<br>
+
Annealing T: 59ºC, elongation time 2.5 minutes.<br>
+
Expected band size: 2275 bp<br>
+
<br>
+
<br>
+
iGEM Cas9<br>
+
Fwd: gtttaactataagaaggagatatacatatgGATAAGAAATACTCAATAGGCTTAGATATC<br>
+
Rev: gccgctctattagtgatggtggtgatgatgGTCACCTCCTAGCTGACTCAAATC<br>
+
Annealing T: 64ºC, elongation time 2.5 minutes.<br>
+
Expected band size: 4088 bp<br>
+
<br>
+
<br>
+
dCas9 & Ala840TAG:<br>
+
Fwd: gtttaactataagaaggagatatacatatgGATAAGAAATACTCAATAGGCTTAGCTATC<br>
+
Rev: gccgctctattagtgatggtggtgatgatgGTCACCTCCTAGCTGACTCAAATC<br>
+
Annealing T: 66ºC, elongation time 2.5 minutes.<br>
+
Expected band size: 4088 bp
+
<br>
+
<br>
+
Ala10TAG:<br>
+
Fwd: gtttaactataagaaggagatatacatatgGATAAGAAATACTCAATAGGCTTATAGATC<br>
+
Rev: gccgctctattagtgatggtggtgatgatgGTCACCTCCTAGCTGACTCAAATC<br>
+
Annealing T: 64ºC, elongation time 2.5 minutes.<br>
+
Expected band size: 4088 bp<br>
+
<br>
+
<br>
+
Positive control: Some ~2000 bp thing from Thomas with iGEM prefix and suffix primers.
+
<br>
+
<br>
+
Fragments were checked by <a href="https://static.igem.org/mediawiki/2016/0/0a/T--Wageningen_UR--Gel_Electrophoresis.pdf">gel electrophoresis</a>. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/2/29/T--Wageningen_UR--Belnote_cas_expr_pcr.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
PCR products were cleaned with Zymo kit (hyperlink), eluted in water and assembled by <a href="https://static.igem.org/mediawiki/2016/1/18/T--Wageningen_UR--HiFi_Gibson_Assembly.pdf">Gibson Assembly</a>. A vector : insert ratio of 1 : 2 was used, with 100 ng vector. 1 uL of Gibson mixtures were transformed in 25uL commercial competent cells (NEB) according to the accompanying protocol and plated on LB plates with kanamycin.
+
<br>
+
<br>
+
Colonies that came up were verified with <a href="https://static.igem.org/mediawiki/2016/f/f8/T--Wageningen_UR--Colony_PCR.pdf">Colony PCR</a>.<br>
+
Primers that were used: <br>
+
Fwd: TTGAAGGGTAGTCCAGAAG<br>
+
Rev: CATATGTATATCTCCTTCTTATAGTTAAAC<br>
+
Annealing T: 46ºC, elongation time 3 minutes. <br>
+
Expected band size: 2647 bp. <br>
+
<br>
+
PCRs were verified using <a href="https://static.igem.org/mediawiki/2016/0/0a/T--Wageningen_UR--Gel_Electrophoresis.pdf">gel electrophoresis</a>. It seemed that there were a lot of positive colonies. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/6/6b/T--Wageningen_UR--Belnote_cas_expr_colPCR.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
Correct clones were confirmed by sequencing.
+
<br>
+
<br>
+
<b>Collection and construction of pEVOL plasmids</b><br>
+
pEVOL-Asp was constructed according to the <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas2">yeast assembly protocol</a>.
+
<br>
+
<br>
+
pEVOL-pAzF<br>
+
Fwd: ACTAGTGCATGCTCGAGCAG<br>
+
Rev: CCTCCTGTTAGCCCAAAAAAACGGGTATG<br>
+
Annealing T: 68ºC, elongation time 2 minutes. <br>
+
Expected band size: ~3320 bp<br>
+
<br>
+
pYES2<br>
+
Fwd: gagcaggcttttttactagtACTCTTCCTTTTTCAATGGG<br>
+
Rev: aaagcaaattcgaccctgagctgctcgagcatgcactagtAAATATTTGCTTATACAATCTTCC<br>
+
Annealing T: 56ºC, elongation time 2 minutes. <br>
+
Expected band size: 2667 bp<br>
+
<br>
+
gBlock1<br>
+
Fwd: gagcaggcttttttactagtACTCTTCCTTTTTCAATGGG<br>
+
Rev: ACAGGGTATTGCTTACGTACCAACTC<br>
+
Annealing T: 66ºC, elongation time 2 minutes. <br>
+
Expected band size: 1203 bp<br>
+
<br>
+
gBlock2<br>
+
Fwd: TTGCTCATGAAATTGAGTTGGTACGTAAG<br>
+
Rev: CCCATTGAAAAAGGAAGAGTACTAG<br>
+
Annealing T: 64ºC, elongation time 2minutes. <br>
+
Expected band size: 1230 bp<br>
+
<br>
+
PCR products were verified using <a href="https://static.igem.org/mediawiki/2016/0/0a/T--Wageningen_UR--Gel_Electrophoresis.pdf">gel electrophoresis</a>. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/6/6c/T--Wageningen_UR--Belnote_pevol_asp_pcr.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
PCR products were cleaned up using the Zymo kit (hyperlink).
+
<br>
+
<br>
+
Then, yeast assembly was performed using the <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas2">protocol</a>, with competent yeast cells received from a supervisor.
+
<br>
+
From the resulting colonies, 6 were picked for <a href="https://static.igem.org/mediawiki/2016/b/bd/T--Wageningen_UR--MiniPrep.pdf">plasmid isolation</a>. Only 3 of them had some plasmid yield, which were checked for correct assembly using OneTaq <a href="https://static.igem.org/mediawiki/2016/2/27/T--Wageningen_UR--Polymerase_Chain_Reaction.pdf">PCR</a>.
+
<br>
+
<br>
+
PCR reactions that were performed:
+
<br>
+
<br>
+
pEVOL fwd and gBlock 1 rev primers, annealing T: 56ºC, elongation 4 minutes. Expected fragment size: 4469 bp. <br>
+
gBlock 1 fwd and gBlock 2 rev primers, annealing T: 50ºC, elongation 4 minutes. Expected fragment size: 2318 bp. <br>
+
<br>
+
<a href="https://static.igem.org/mediawiki/2016/0/0a/T--Wageningen_UR--Gel_Electrophoresis.pdf">gel electrophoresis</a> reveiled fragments of the right size for colony 2 and 3. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/c/ca/T--Wageningen_UR--Belnote_pevol_asp_colPCR.JPG">
+
</figure><br/>
+
 
+
<p><br>
+
Eventually, the plasmid from colony 3 was <a href="https://static.igem.org/mediawiki/2016/0/00/T--Wageningen_UR--Preparing_and_transforming_DH5%CE%B1_electrocompetent_cells.pdf">transformed</a> successfully in <i>E. coli</i>, <a href="https://static.igem.org/mediawiki/2016/b/bd/T--Wageningen_UR--MiniPrep.pdf">miniprepped</a> and sent for sequencing. The following mutations were present: Tryp156Cys, Gly321Val and Gly525Cys. Because there was no time to check other clones, I continued with this plasmid anyways.
+
<br>
+
<br>
+
<b>Expression of Cas9-expresso constructs in C321ΔA</b><br>
+
Both the acquired Cas9-expresso constructs as well as pEVOL-Asp were transformed into C321ΔA as described in <i>Lajoie et al.</i> (2013)<sup><a href="#bp1" id="refbp1">1</a></sup>.
+
</section>
+
 
+
<section id="6">
+
<h1><b>Sept 1 - Okt 10</b></h1>
+
<p>
+
<b>Expression of Cas9-expresso constructs in C321ΔA</b><br>
+
An expression experiment was performed with 3 ml cultures induced overnight with rhamnose, arabinose and synthetic amino acids when applicable. This yielded no visible Cas9 bands.
+
The same happened when 5 ml cultures where induced for 4 hours. What worked, was the <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas5">protocol</a> with bigger volumes followed by Ni-NTA purification (the majority of the actual work with the FPLC was performed by a supervisor)
+
<br>
+
<br>
+
First, samples were purified as described, but with addition of DNAse. This gave good yields, but DNAse remained in the purified fractions as was later found out during <i>in vitro</i> Cas9 assays. However, without DNAse also a good yield was obtained. </p>
+
 
+
<figure>
+
<a href="https://static.igem.org/mediawiki/2016/1/1f/T--Wageningen_UR--Belnote_fplc_turned.JPG"><img src="https://static.igem.org/mediawiki/2016/1/1f/T--Wageningen_UR--Belnote_fplc_turned.JPG" width="700"></a>
+
<figcaption>Click the figure for the full-resolution image.<figcaption>
+
</figure><br/>
+
</section>
+
 
+
<p>
+
The green line indicates the amount of His buffer B that is passed through the column. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/9/94/T--Wageningen_UR--Bel_overview_SDS.JPG">
+
<figcaption>Figure 2. SDS-PAGE of fractions after FPLC purification of Cas9 variants. Cas9 = 156 kDa. Red boxes indicate 150 kDa band of the ladder, red arrows indicates bands of the correct size corresponding to Cas9. The black arrow indicates elution with increasing concentration of Imidazole. a) Cas9. b) dCas9. c) dCas9-Ala10BipA. d) dCas9-Ala10Asp. e)dCas9-Ala10TAG, no synthetic amino acid. Ladder: Precision Plus protein ladder (Bio-Rad). CFE = Cell Free Extract. </figcaption>
+
</figure><br/>
+
 
+
<p>
+
<br>
+
<b><i>in vitro</i> Cas9 cleaving assays</b><br>
+
Assays were performed with all produced guide RNAs, according to the <a href="https://2016.igem.org/Team:Wageningen_UR/Experiments#cas6">protocol</a>. </p>
+
 
+
<figure>
+
<img src="https://static.igem.org/mediawiki/2016/2/24/T--Wageningen_UR--Bel_invitro_temp.JPG">
+
<figcaption>Figure 3. <i>in vitro</i> Cas9 activity assays with Cas9, dCas9 and dCas9-Ala10Asp. Substrate for cleaving is a PCR product including the gene encoding RFP, which is targeted at the N-terminal side, both on the template strand (guideRNA 2 and 4) and the non-template strand (gRNA 1). Size of the uncleaved PCR product is 4140 bp, cleaving generates a 3100 bp and a 1040 bp fragment. Ladder: 1kb (NEB).</figcaption>
+
</figure><br/>
+
 
+
</section>
+
 
+
<section id="7">
+
<h1><b>References</b></h1>
+
 
+
<a id="bp1" href=http://science.sciencemag.org/content/342/6156/357>1.</a> ajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H. R., Haimovich, A. D., Kuznetsov, G., ... & Rohland, N. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357-360.
+
<a href="#refbp1" title="Jump back to footnote 1 in the text.">↩</a>
+
<br><br>
+
 
+
</section>
+
  
 +
<h2><b>October 3rd – Project Presentation</b></h2>
 +
<p> On October the 3rd, we organized a lunch lecture at the university to present our project to researchers, beekeepers, students and other interested people. Quite some people were attending and a lot of questions were asked. We are happy about getting to practice our presentation in front of an audience, as well al learning what parts of our story where still unclear. Furthermore, we hope we enthused other students who might participate in next year’s iGEM team!
 +
</p>
 
</html>
 
</html>
{{Wageningen_UR/footer}}
 

Revision as of 15:01, 17 October 2016

Wageningen UR iGEM 2016

 

March 23rd – Multi-Stakeholder Dialogue

The Role of Synthetic Biology in Energy Transition

To broaden our horizon and understanding of the possibilities of synthetic biology, we joined a multi-stakeholder dialogue that was organized to discuss the role of synthetic biology in energy transition, by the Athena institute and the Rathenau institute. There is a general belief that something needs to change about the current Dutch energy supply, and during this dialogue we brainstormed about how synthetic biology could be of help achieving this. In a guided way, we generated ideas about applications of synthetic biology for energy supply, starting from future society-scenario’s created the day before. The nice thing about this approach was that we learned about the perspectives of people with different backgrounds, ranging from scientists to business people. It felt like we all started on the same page. The ideas that were most appealing were later worked out in different groups. Lisa and I worked on a scenario, “the green human”, where humans would evolve together with plants to be able to rely on photosynthesis for energy. Of course this scenario is very futuristic, but in our opinion it exposes a very important aspect of both energy transition and synthetic biology: that the public should be involved, to be more conscious about energy use and more engaged with technological advances in the field of synthetic biology.

April – Meetings with DAE

As mentioned in the collaboration section, we designed our project with the help of students from the Design Academy Eindhoven. Apart from collaborating with them on designing the future end product BeeT, they helped us think about our project and its connection with society. We met with them twice: The first time, we visited them and took part in a design workshop. We talked about our ideas for our iGEM projects - at that time there were still a lot of ideas on the table. The students helped us realize what other “non-science” people think about synthetic biology. The second meeting was a lab day for them to see what our everyday life in the lab looks like. It was fun sharing our knowledge with them. On this day, we especially realized what kind of prejudices a lot of people have towards GMOs. At the end of the day, we learned what aspects we should pay special attention to when presenting our project to the broad public. Moreover, we were able to convince the students that synthetic biology is in fact something good rather than something threatening.

The movie Leif Czakai (DAE student) made from us, making baklava.

May 21st – National Meetup

On 21/05/2016 we organised the meet-up of all interested Dutch iGEM teams. Four teams took part: Groningen, Leiden, Eindhoven, and us. The meeting was divided in two parts. The first part consisted of every team presenting their project ideas with a brief discussion afterwards. After lunch the brainstorm session started. We divided the group in 4 random subgroups. The groups got to know eachother a bit, and then brainstormed on whatever iGEM related topic they liked. Some groups decided to talk about what team could collaborate with what other team, some teams decided to think about ways to connect in a better way, and some teams thought about how to make iGEM more popular in the Netherlands. After some small presentations of the brainstorm sessions’ results, we discussed several ideas. The outcome of the discussions was:
• a list with contact details and team roles of every Dutch team member has been obtained
• the notion of several collaboration possibilities
• ideas about shared sponsoring and funding
When the discussions were over, the official part came to an end, but some teams ended up ordering pizza and playing board games together. All in all, we feel the day was a success not only for the progress of our projects, but also for getting to know and connecting with our fellow Dutch iGEM teams.

June 24th & 25th – Synenergene Forum

In search for the right conditions for responsible research and innovation, this conference was organized by the Rathenau institute (hyperlink) for all relevant stakeholders when it comes to the future of synthetic biology. Scientists, business people, policymakers and civil society organizations attended, but it was also open for the general public. The promises and pitfalls of synthetic biology were considered from a societal perspective during pitches, presentations, workshops and debates. We attended a workshop about connecting synthetic biology with culture and religion, a link we had not thought about before. During small information rounds, we had the opportunity to talk to experts in the field. We also joined a plenary discussion about freedom and security in an age of synthetic biology. We enjoyed hearing so many different opinions and having the opportunity to talk to experts from all over the world.

September 20th – RIVM meeting

Making the Safe by design movie

In contrast to many other synthetic biology applications, BeeT is intended to be used outside the lab, in beehives, which is in close contact with nature. To get a better idea of the viability of our idea, and what it is like to request permission to use an engineered microorganism outside of the lab, we looked at the questions that are asked in such a request (link to website of RIVM) and discuss some that peaked our interest in the movie. Some interesting questions we had not thought about, such as “how can a genetically modified microorganism be recognized among natural relatives?”. Another interesting aspect of the application of synthetic biology is responsibility: if our engineered bacterium would be commercialized, who would be responsible for any adverse effects? In our opinion, scientists are responsible for thoroughly and objectively investigating and reporting possible risks. Companies in turn should monitor whether the technology is sufficiently researched. Companies should also make sure that user manuals are correct and clear to understand, even to people with little experience with comparable technology. Users of the product, the beekeepers in our case, are responsible for using the product in the intended way only and following the user manual provided with the product. Our movie ends with some open questions regarding safe by design, both in general and within our project. Examples are “what should be done to actually realize the insights that are gained by safe by design?” and “should there be more focus on the positive aspects of using a genetically modified microorganism when judging a permission request?”. We don’t know the answer to these questions, but think they are important nevertheless.

The movie we made in collaboration with the RIVM.

Meeting

Concluding our collaboration with the RIVM, some members of our team, as well as the other Dutch iGEM teams attended a meeting organized by the RIVM and the Rathenau institute, with the theme: “veilig verder met synthetische biologie”, or “making safe progress in synthetic biology”. During this meeting, researchers, policymakers and policy advisors brainstormed, discussed and presented their ideas about how synthetic biology should be handled in the future. We gave a short presentation about the topic of our project and how we integrated “safe by design” through toxin specificity, controlled expression of the toxin and confinement of our product to the beehive. During a short information market, we got to talk more in depth with people who were interested, presented the movie we made for the RIVM and the Resource from the future that was made in collaboration with Synenergene. There were short “break-out sessions” where safety of previous iGEM projects was discussed in small groups, and at the same time two artists made a huge and beautiful drawing of everything that was discussed during the day. We learned a lot from the interesting talks about further handling safety in synthetic biology both from a political and a societal point of view. An interesting point that comes to mind was made by Sabine Roeser (professor of Ethics, TU Delft), about how feelings of the public concerning synthetic biology can be a useful source of questions on the ethical aspects of our work. She also mentioned how art can be used to connect people to synthetic biology. In our project, we tried to achieve this through collaborating with the Design Academy Eindhoven (hyperlink). Besides, it was nice to get to know more about how the other Dutch iGEM teams handled the RIVM assignment. We thank the RIVM and the Rathenau institute for inviting us, it was an inspiring day!

October 3rd – Project Presentation

On October the 3rd, we organized a lunch lecture at the university to present our project to researchers, beekeepers, students and other interested people. Quite some people were attending and a lot of questions were asked. We are happy about getting to practice our presentation in front of an audience, as well al learning what parts of our story where still unclear. Furthermore, we hope we enthused other students who might participate in next year’s iGEM team!