Cas9 & Cpf1 secretion
and activity
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<div class="text">InterLab study participation</div> | <div class="text">InterLab study participation</div> | ||
<div class="desc">Results and details of our measurements for the iGEM 2017 InterLab Study.</div> | <div class="desc">Results and details of our measurements for the iGEM 2017 InterLab Study.</div> | ||
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<div class="page-heading">MESA construct replication</div> | <div class="page-heading">MESA construct replication</div> | ||
| − | The architecture we use for OUTCASST is inspired by the Modular Extracellular Sensor Architecture (MESA) <i class="ref" data- | + | The architecture we use for OUTCASST is inspired by the Modular Extracellular Sensor Architecture (MESA) <i class="ref" data-id="1">1</i> <i class="ref" data-id="2">2</i>. Because of this, MESA first needed to be replicated to verify whether the final product could work as intended. |
A successful replication is required for OUTCASST to work, and to provide data that we can use to compare and correct models of the system. | A successful replication is required for OUTCASST to work, and to provide data that we can use to compare and correct models of the system. | ||
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It can be used as a sensor as it has two different protein chains with, on the extracellular region, an element to cause dimerization and, on the intracellular region, a combination of a protease on one chain and a transcription factor on the other. The transcription factor can be cleaved off by the protease when the two chains dimerize, either through a ligand or randomly. Subsequently, the transcription factor travels to the nucleus where it induces transcription of a reporter (Figure 1). | It can be used as a sensor as it has two different protein chains with, on the extracellular region, an element to cause dimerization and, on the intracellular region, a combination of a protease on one chain and a transcription factor on the other. The transcription factor can be cleaved off by the protease when the two chains dimerize, either through a ligand or randomly. Subsequently, the transcription factor travels to the nucleus where it induces transcription of a reporter (Figure 1). | ||
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| − | <b>Figure 1. | + | <span class="text-figure"> |
| + | <b>Figure 1. The MESA signalling pathway.</b> The MESA cell signalling pathway consists of a target chain (TC), a protease chain (PC) and a ligand which can bind both the extracellular domains. The TC has a transcription factor on the intracellular part of the protein, while the PC has a protease which can cleave off the aforementioned transcription factor. The ligand, in this case VEGF, binds both chains, which allows the protease to be close enough to do this. The released transcription factor subsequently travels to the nucleus to induce a reporter gene. | ||
| + | Image modified from <i class="ref" data-id="3">1</i>. | ||
| + | </span> | ||
<br><br> | <br><br> | ||
The MESA constructs that form the basis of OUTCASST, and therefore need to be verified, are V2-MESA-35F-M-tTA and V2-MESA-35F-TEV. | The MESA constructs that form the basis of OUTCASST, and therefore need to be verified, are V2-MESA-35F-M-tTA and V2-MESA-35F-TEV. | ||
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<h2 class="subhead" id="subhead-3">Materials</h2> | <h2 class="subhead" id="subhead-3">Materials</h2> | ||
<ul> | <ul> | ||
| − | <li />V2-MESA-35F-M-tTA | + | <li />V2-MESA-35F-M-tTA <a href="https://www.addgene.org/84502/" class="url_external"></a> |
<li />V2-MESA-35F-TEV (https://www.addgene.org/84503/) | <li />V2-MESA-35F-TEV (https://www.addgene.org/84503/) | ||
<li />pL3-TRE-LucGFP-2L (https://www.addgene.org/11685/) | <li />pL3-TRE-LucGFP-2L (https://www.addgene.org/11685/) | ||
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Revision as of 18:52, 31 October 2017
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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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