Cas9 & Cpf1 secretion
and activity
Difference between revisions of "Template:Team:Utrecht/MainBody"
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The OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them. | The OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them. | ||
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| − | As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration | + | As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration <i class="ref" data-id="1">1</i>. This way the shelf life of our tool can also be prolonged. To prevent the risk of our GMO getting out in the environment, several mechanisms and kill-switches will be incorporated in the cells, so they can only survive in our closed box system, in their resurgent state. |
This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, e.g. an amino acid, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance. | This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, e.g. an amino acid, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance. | ||
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We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. | We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. | ||
We want to take this information into account to design the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the <i>T. cruzi</i> parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist). | We want to take this information into account to design the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the <i>T. cruzi</i> parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist). | ||
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| + | <h2 class="subhead" id="subhead-4">References</h2> | ||
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| + | <ol class="references"> | ||
| + | <li data-title="Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term…" data-author="Watanabe K, Imanishi S, Akiduki G, Cornette R, Okuda T." data-link="https://doi.org/10.1016/j.cryobiol.2016.05.006" /> Watanabe K, Imanishi S, Akiduki G, Cornette R, Okuda T. Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration. Cryobiology. 2016 Aug 31;73(1):93-8. <a target=_BLANK href="https://doi.org/10.1016/j.cryobiol.2016.05.006" class="url_external"></a> | ||
| + | </ol> | ||
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| + | <span id="tooltip-1"></span> | ||
--></script> | --></script> | ||
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<h2 class="subhead" id="subhead-2">iGEM Utrecht: an introduction</h2> | <h2 class="subhead" id="subhead-2">iGEM Utrecht: an introduction</h2> | ||
| − | <video | + | <video controls> |
| − | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/b/bb/UuHPOutreach1.mp4" type='video/mp4'/> |
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<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
| + | <div class="description">What does iGEM mean and what’s so fun about it? What are the biggest challenges faced during the experiments. In this movie we explain it all.</div> | ||
</div> | </div> | ||
<div class="outreach-video right"> | <div class="outreach-video right"> | ||
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<h2 class="subhead" id="subhead-2">Meet the team</h2> | <h2 class="subhead" id="subhead-2">Meet the team</h2> | ||
| − | <video | + | <video controls> |
| − | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/6/68/UuHPOutreach2.mp4" type='video/mp4'/> |
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<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
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| + | <div class="description">During the formation of the team, a lot of attention is paid to diversity of the members and their background. In this movie some of the members introduce themselves and explain why they participate in this competition.</div> | ||
</div> | </div> | ||
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<h2 class="subhead" id="subhead-2">The problem; the detection of infectious disease has to be improved</h2> | <h2 class="subhead" id="subhead-2">The problem; the detection of infectious disease has to be improved</h2> | ||
| − | <video poster="" controls> | + | <video poster="https://static.igem.org/mediawiki/2017/6/6c/Uu-outreach-video3-poster.png" controls> |
| − | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/b/bb/UuHPOutreach3.mp4" type='video/mp4'/> |
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<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
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| + | <div class="description">For which problem do we find a solution in our research? The concept of the problem is explained in this draw-my-life video.</div> | ||
</div> | </div> | ||
<div class="outreach-video right"> | <div class="outreach-video right"> | ||
| − | <h2 class="subhead" id="subhead-2"> | + | <h2 class="subhead" id="subhead-2">Labsafety for Dummies</h2> |
| − | <video | + | <video controls> |
| − | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/4/4c/UuHPOutreach4.mp4" type='video/mp4'/> |
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<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
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| + | <div class="description">The lab can be a treacherous environment. One tiny mistake, and BOOM! We answer important questions about how to stay safe in the lab.</div> | ||
</div> | </div> | ||
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<div style="clear: both;"></div> | <div style="clear: both;"></div> | ||
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| + | <div style="clear: both;"> | ||
| + | For a link to the special on our team on the website of de Kennis van Nu, <a target=_BLANK href="https://dekennisvannu.nl/site/special/iGEM-2017-studenten-ontwerpen-nieuw-leven/111#!/" class="url_external">click here</a>. | ||
| + | </div> | ||
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.outreach-video>video { width: 100%; } | .outreach-video>video { width: 100%; } | ||
.outreach-video>h2 { font-size: 17px; color: black; font-weight: bold; padding-bottom: 10px; border-bottom: 2px solid #f6f6f6; } | .outreach-video>h2 { font-size: 17px; color: black; font-weight: bold; padding-bottom: 10px; border-bottom: 2px solid #f6f6f6; } | ||
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| + | .outreach-video .description { font-size: 12px; line-height: 20px; display: inline-block; } | ||
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2: "Toolkit design solutions", | 2: "Toolkit design solutions", | ||
3: "Additional considerations", | 3: "Additional considerations", | ||
| + | 4: "References" | ||
}, | }, | ||
"outreach" : {}, | "outreach" : {}, | ||
Revision as of 16:47, 1 November 2017
<!DOCTYPE html>
Comparison of endonuclease activity for Cas9 and Cpf1 that has been produced in, and excreted by, HEK293 cells.
MESA two-component system replication
Details on the MESA two-component system, explanation of its relation to our design and the results of its reproduction.
OUTCASST system production
Detailed explanation of the OUTCASST mechanism, experimental progress and technical prospects.
Modeling and
mathematics
mathematics
Ordinary differential equations, cellular automaton and an object based model for optimal linker-length estimation.
InterLab study participation
Results and details of our measurements for the iGEM 2017 InterLab Study.
Stakeholders & opinions
Interviews and dialogues with stakeholders, potential users, third parties and experts relating to pathogen detection or DNA-based diagnostics.
Risks & safety-issues
Implications and design considerations relating to safety in the usage and implementation of OUTCASST as a diagnostics tool.
Design & integration
OUTCASST toolkit and product design with factors such as bio-safety and user-friendliness taken into account.
Outreach
Videos we made for the dutch public, together with 'de Kennis van Nu'.
Meet our team
About us, our interests and roles in the team and our supervisors.
Sponsors
A listing of our sponsors, how they assisted us and our gratitude for their assistance.
Collaborations
Read about our exchanges with other iGEM teams and government agencies.
Achievements
A short description of all that we have achieved during our participation in the iGEM.
Attributions
A thank-you for everyone that assited us, both in and outside the lab.
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