Cas9 & Cpf1 secretion
and activity
Difference between revisions of "Template:Team:Utrecht/MainBody"
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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 <a href="https://www.addgene.org/84502/" class="url_external"></a> | + | <li />V2-MESA-35F-M-tTA <a target=_BLANK href="https://www.addgene.org/84502/" class="url_external"></a> |
| − | <li />V2-MESA-35F-TEV | + | <li />V2-MESA-35F-TEV <a target=_BLANK href="https://www.addgene.org/84503/" class="url_external"></a> |
| − | <li />pL3-TRE-LucGFP-2L | + | <li />pL3-TRE-LucGFP-2L <a target=_BLANK href="https://www.addgene.org/11685/" class="url_external"></a> |
| − | <li />Cre reporter | + | <li />Cre reporter <a target=_BLANK href="https://www.addgene.org/62732/" class="url_external"></a> |
| − | <li />pBI-MCS-EYFP | + | <li />pBI-MCS-EYFP <a target=_BLANK href="http://www.addgene.org/58855/" class="url_external"></a> |
<li />pSLQ-Set1-BFP (not on addgene) | <li />pSLQ-Set1-BFP (not on addgene) | ||
| − | <li />VEGF-164 | + | <li />VEGF-164 (cat.: 583102, biolegend) |
</ul> | </ul> | ||
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The results of the flow cytometry analysis are shown in table 1. The GFP and YFP reporter plasmid concentrations for transfection were varied between 250 ng and 350 ng per well. After adding VEGF, GFP signal increased in all samples except for the sample containing 250 ng GFP, which showed a decrease. The cells that were transfected with a GFP concentration of 350 ng showed the greatest increase in GFP activity (9.2%). In figure 2 the FACS plots for 350 ng GFP are shown. | The results of the flow cytometry analysis are shown in table 1. The GFP and YFP reporter plasmid concentrations for transfection were varied between 250 ng and 350 ng per well. After adding VEGF, GFP signal increased in all samples except for the sample containing 250 ng GFP, which showed a decrease. The cells that were transfected with a GFP concentration of 350 ng showed the greatest increase in GFP activity (9.2%). In figure 2 the FACS plots for 350 ng GFP are shown. | ||
<br><br> | <br><br> | ||
| − | <b>Table 1. | + | <span class="text-figure"> |
| + | <b>Table 1. Heatmap of the flow cytometry data.</b> The percentages shown is the relative difference of fluorescence compared to control. The colour scale has been set with a minimum of -19.4%, a midpoint of 0% and maximum of 27.1%. Overall, signal induction appears to be more efficient with the YFP plasmid, but the data is inconsistent. | ||
| + | </span> | ||
<br> | <br> | ||
| − | <img width="600" src="https://static.igem.org/mediawiki/2017/7/70/Uufacsheatmap.png"> | + | <center><img style="margin-top: 10px;" width="600" src="https://static.igem.org/mediawiki/2017/7/70/Uufacsheatmap.png"></center> |
<br> | <br> | ||
<br> | <br> | ||
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<br><br> | <br><br> | ||
| − | <img | + | <center><img style="margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/0/0d/Uumesafig2_merge.png"></center> |
| − | < | + | <span class="text-figure"> |
| − | <b>Figure 2. | + | <b>Figure 2. FACS results for treatment with 350 ng GFP plasmid.</b> (Left) Plot before adding VEGF. (Right) Plot after adding VEGF. [GFP] is GFP activity. |
| + | </span> | ||
<br> | <br> | ||
<br> | <br> | ||
| − | <img | + | <center><img style="margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/5/5c/Uumesafig3_merged.png"></center> |
| − | < | + | <span class="text-figure"> |
| − | <b>Figure 3. | + | <b>Figure 3. FACS results for treatment with 300 ng YFP plasmid, before VEGF was added.</b> (Left) Plot before adding VEGF. (Right) Plot after adding VEGF. [Q2] is YFP activity. |
| + | </span> | ||
<br><br> | <br><br> | ||
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<br><br> | <br><br> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/8/8c/Uumesafig4_merged.png"></center> |
| − | < | + | <span class="text-figure"> |
| − | <b>Figure 4. | + | <b>Figure 4. FACS results for treatment after VEGF addition.</b> (Left) with protease chain. (Right) without protease chain. [Q2] is YFP activity. |
| + | </span> | ||
<br> | <br> | ||
<br> | <br> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/8/8e/Uumesafig5_merged.png"></center> |
| − | < | + | <span class="text-figure"> |
| − | <b>Figure 5.</b> | + | <b>Figure 5. 350 ng GFP.</b> (Left) with protease chain. (Right) without protease chain. [GFP] is GFP activity. |
| + | </span> | ||
<br><br> | <br><br> | ||
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<ol class="references"> | <ol class="references"> | ||
| − | <li data-title="Modular extracellular sensor architecture for engineering mammalian cell-based devices." data-author="Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N." data-link="http://pubs.acs.org/doi/abs/10.1021/sb400128g" />Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N., 2014: Modular extracellular sensor architecture for engineering mammalian cell-based devices. ACS synthetic biology, 3(12), 892-902. <a target=_BLANK href="http://pubs.acs.org/doi/abs/10.1021/sb400128g" class=" | + | <li data-title="Modular extracellular sensor architecture for engineering mammalian cell-based devices." data-author="Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N." data-link="http://pubs.acs.org/doi/abs/10.1021/sb400128g" />Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N., 2014: Modular extracellular sensor architecture for engineering mammalian cell-based devices. ACS synthetic biology, 3(12), 892-902. <a target=_BLANK href="http://pubs.acs.org/doi/abs/10.1021/sb400128g" class="url_external"></a> |
| − | <li data-title="Rewiring human cellular input-output using modular extracellular sensors." data-author="Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N." data-link="http://www.nature.com/nchembio/journal/v13/n2/abs/nchembio.2253.html?foxtrotcallback=true" />Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N. 2017: Rewiring human cellular input-output using modular extracellular sensors. Nature chemical biology, 13(2), 202-209. <a target=_BLANK href="http://www.nature.com/nchembio/journal/v13/n2/abs/nchembio.2253.html?foxtrotcallback=true" class=" | + | <li data-title="Rewiring human cellular input-output using modular extracellular sensors." data-author="Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N." data-link="http://www.nature.com/nchembio/journal/v13/n2/abs/nchembio.2253.html?foxtrotcallback=true" />Schwarz, K. A., Daringer, N. M., Dolberg, T. B., & Leonard, J. N. 2017: Rewiring human cellular input-output using modular extracellular sensors. Nature chemical biology, 13(2), 202-209. <a target=_BLANK href="http://www.nature.com/nchembio/journal/v13/n2/abs/nchembio.2253.html?foxtrotcallback=true" class="url_external"></a> |
</ol> | </ol> | ||
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<b>Cas9 Mutagenesis</b> | <b>Cas9 Mutagenesis</b> | ||
<br> | <br> | ||
| − | The miniprepped sample sent for sequencing and the results of the sequencing showed that the D10A mutation was successfully introduced to Cas9. The sequenced sample was used as a template to create the second mutation. Transformation for this product failed several times. Even with successful transformation, the concentration after miniprep was too low to send for sequencing. For the sake of time, we was decided that the DNA sequence for dCas9 would be ordered from addgene as a whole. The following plasmid, including the mutations of our design, was ordered: https://www.addgene.org/46911/. | + | The miniprepped sample sent for sequencing and the results of the sequencing showed that the D10A mutation was successfully introduced to Cas9. The sequenced sample was used as a template to create the second mutation. Transformation for this product failed several times. Even with successful transformation, the concentration after miniprep was too low to send for sequencing. For the sake of time, we was decided that the DNA sequence for dCas9 would be ordered from addgene as a whole. |
| + | The following plasmid, including the mutations of our design, was ordered: <a target=_BLANK href="https://www.addgene.org/46911/" class="url_external">https://www.addgene.org/46911/</a>. | ||
<br><br> | <br><br> | ||
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<br> | <br> | ||
| + | <div style="float: right; width: 200px; margin-left: 50px;"> | ||
| + | <img style="width: 100%;" src="https://static.igem.org/mediawiki/2017/thumb/7/72/Restriction_digest_dCpf1-tTa_clone_8.png/325px-Restriction_digest_dCpf1-tTa_clone_8.png"> | ||
| + | <br> | ||
| + | <span class="text-figure" style="display: inline-block; line-height: 20px;"> | ||
| + | <b>Figure 1. restriction digest of dCpf1-tTa clone 8 using BamHI and XhoI.</b> Two fragments were expected to be the result of the restriction digest, one fragment of 6635 bp and one of 4792 bp. Only one fragment of approximately 11.000 bp appeared, leading to the conclusion that the clone was not correct. | ||
| + | </span> | ||
| + | </div> | ||
After InFusion and transformation, plasmids were miniprepped and clone 2, 3, 4, 5 and 8 were sent for sequencing. Unfortunately, the sequence data looked chaotic and none of the clones were completely correct. It could have been that the DNA quality was not good enough for sequencing. Of these results, clone 8 looked the most promising and so this one was re-transformed, maxiprepped and examined with restriction digestion. | After InFusion and transformation, plasmids were miniprepped and clone 2, 3, 4, 5 and 8 were sent for sequencing. Unfortunately, the sequence data looked chaotic and none of the clones were completely correct. It could have been that the DNA quality was not good enough for sequencing. Of these results, clone 8 looked the most promising and so this one was re-transformed, maxiprepped and examined with restriction digestion. | ||
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Sequencing results showed that there was likely to be a mistake in the backbones of these clones that could be fixed through a PCR treatment for the backbone section with the error. This fragment could then be replaced in the construct through restriction and InFusion. | Sequencing results showed that there was likely to be a mistake in the backbones of these clones that could be fixed through a PCR treatment for the backbone section with the error. This fragment could then be replaced in the construct through restriction and InFusion. | ||
| + | |||
| + | <center><img style="margin-top: 10px;" width="600" src="https://static.igem.org/mediawiki/2017/thumb/8/80/Restriction_digest_dCpf1-tTa_attempt_2.png/800px-Restriction_digest_dCpf1-tTa_attempt_2.png"></center> | ||
| + | <span class="text-figure"> | ||
| + | <b>Figure 2. restriction digest of dCpf1-tTa clone 2, 3, 4 and 8 using BamHI and NcoI.</b> Again, none of the samples were correct. | ||
| + | </span> | ||
<br><br> | <br><br> | ||
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In conclusion, for minimization of false positive results, the cleavage rate should result in a cleavage duration that is near the half-life of the uncleaved complex between target-chain, protease-chain and substrate. Increasing the cleavage rate would not contribute to sensitivity and merely decrease precision. | In conclusion, for minimization of false positive results, the cleavage rate should result in a cleavage duration that is near the half-life of the uncleaved complex between target-chain, protease-chain and substrate. Increasing the cleavage rate would not contribute to sensitivity and merely decrease precision. | ||
<br><br> | <br><br> | ||
| − | A full work-out of this demonstration in mathematica notebook can be found <a target=_BLANK href="https://drive.google.com/drive/folders/0B_qbow6tESp8b2FNb3pMa1psLTA">here</a>. | + | A full work-out of this demonstration in mathematica notebook can be found <a target=_BLANK href="https://drive.google.com/drive/folders/0B_qbow6tESp8b2FNb3pMa1psLTA" class="url_external">here</a>. |
<br><br> | <br><br> | ||
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<h2 class="subhead" id="subhead-3">Materials</h2> | <h2 class="subhead" id="subhead-3">Materials</h2> | ||
<ul> | <ul> | ||
| − | <li />InterLab Parts and Measurement Kit | + | <li />InterLab Parts and Measurement Kit <a target=_BLANK href="http://parts.igem.org/Help:2017_DNA_Distribution#Measurement_Kit" class="url_external"></a> |
<li />Plate reader: Biotek Synergy HT | <li />Plate reader: Biotek Synergy HT | ||
| − | <li /><i>E. coli</i> | + | <li /><i>E. coli DH5-alpha</i> cells |
</ul> | </ul> | ||
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<h2 class="subhead" id="subhead-4">Methods</h2> | <h2 class="subhead" id="subhead-4">Methods</h2> | ||
<ul> | <ul> | ||
| − | <li />Protocol for the transformation of competent <i>E. coli</i>: single tube transformation protocol | + | <li />Protocol for the transformation of competent <i>E. coli</i>: single tube transformation protocol <a target=_BLANK href="http://parts.igem.org/Help:Protocols/Transformation" class="url_external"></a> |
| − | <li />Protocol for the plate reader | + | <li />Protocol for the plate reader <a target=_BLANK href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf" class="pdf pdf-inline"></a> |
</ul> | </ul> | ||
<br> | <br> | ||
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<br> | <br> | ||
| − | <img width="600" src="https://static.igem.org/mediawiki/2017/6/60/Uuinterlab_figure1.png"> | + | <center><img style="margin-top: 25px;" width="600" src="https://static.igem.org/mediawiki/2017/6/60/Uuinterlab_figure1.png"></center> |
| − | < | + | <span class="text-figure"> |
<b>Figure 1.</b> The relative fluorescence per cell over time for the negative and the positive controls and the six test devices of the diluted cultures. | <b>Figure 1.</b> The relative fluorescence per cell over time for the negative and the positive controls and the six test devices of the diluted cultures. | ||
| + | </span> | ||
<br> | <br> | ||
<br> | <br> | ||
| − | <img width="600" src="https://static.igem.org/mediawiki/2017/0/03/Uuinterlab_figure2.png"> | + | |
| − | < | + | <center><img style="margin-top: 25px;" width="600" src="https://static.igem.org/mediawiki/2017/0/03/Uuinterlab_figure2.png"></center> |
| + | <span class="text-figure"> | ||
<b>Figure 2.</b> The relative fluorescence per cell over time for the negative and the positive controls and test device 2 till 6 of the diluted cultures. | <b>Figure 2.</b> The relative fluorescence per cell over time for the negative and the positive controls and test device 2 till 6 of the diluted cultures. | ||
| + | </span> | ||
<br><br> | <br><br> | ||
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.text-figure { font-size: 11px; } | .text-figure { font-size: 11px; } | ||
| − | .url_external { background: url(https://static.igem.org/mediawiki/2017/0/0f/Uu_url_external.png) center right no-repeat; padding-right: 15px; } | + | .url_external { background: url(https://static.igem.org/mediawiki/2017/0/0f/Uu_url_external.png) center right no-repeat; height: 13px; padding-right: 15px; } |
#modal-overlay | #modal-overlay | ||
Revision as of 20:39, 31 October 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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