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<span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_0" href="https://2016.igem.org/Team:Tsinghua">Home</a></span> | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_0" href="https://2016.igem.org/Team:Tsinghua">Home</a></span> | ||
<ul class="subs"> | <ul class="subs"> | ||
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<li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_0" href="https://2016.igem.org/Team:Tsinghua/Team">Team members</a></li> | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_0" href="https://2016.igem.org/Team:Tsinghua/Team">Team members</a></li> | ||
− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_1" href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_1" href="https://2016.igem.org/Team:Tsinghua/Attributions">Attributions</a></li> |
− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_2" href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_2" href="https://2016.igem.org/Team:Tsinghua/Acknowledgement">Acknowledgement</a></li> |
− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_3" href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_3" href="https://2016.igem.org/Team:Tsinghua/Acknowledgement#tag_sponsorship">Sponsorship</a></li> |
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− | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_2" href="https://2016.igem.org/Team:Tsinghua/ | + | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_2" href="https://2016.igem.org/Team:Tsinghua/Description">Project</a></span> |
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− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_4" href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_4" href="https://2016.igem.org/Team:Tsinghua/Notebook">Notebook</a></li> |
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+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_5" href="https://2016.igem.org/Team:Tsinghua/Safety">Safety</a></li> | ||
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<span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_3" href="https://2016.igem.org/Team:Tsinghua/Parts">Parts</a></span> | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_3" href="https://2016.igem.org/Team:Tsinghua/Parts">Parts</a></span> | ||
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− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_2" href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_2" href="https://2016.igem.org/Team:Tsinghua/Composite_Part">Composite Parts</a></li> |
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− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_1" href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_1" href="https://2016.igem.org/Team:Tsinghua/Human_Practices#tag_engagement">Public Engagement</a></li> |
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+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_1" href="https://2016.igem.org/Team:Tsinghua/Community_meetup">Community Meetup</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_2" href="https://2016.igem.org/Team:Tsinghua/Integrated_Practices">Integrated Practices</a></li> | ||
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<span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_4" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist">Medal Checklist</a></span> | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_4" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist">Medal Checklist</a></span> | ||
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− | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_0" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#bronze | + | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_0" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#bronze">Bronze</a></li> |
<li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_1" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#silver">Silver</a></li> | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_1" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#silver">Silver</a></li> | ||
<li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_2" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#gold">Gold</a></li> | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_2" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#gold">Gold</a></li> | ||
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<li><a href="https://2016.igem.org/Team:Tsinghua">Home</a></li> | <li><a href="https://2016.igem.org/Team:Tsinghua">Home</a></li> | ||
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− | <li><a href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a href="https://2016.igem.org/Team:Tsinghua/Description">Project</a></li> |
<li><a href="https://2016.igem.org/Team:Tsinghua/Parts">Parts</a></li> | <li><a href="https://2016.igem.org/Team:Tsinghua/Parts">Parts</a></li> | ||
− | <li><a href="https://2016.igem.org/Team:Tsinghua/ | + | <li><a href="https://2016.igem.org/Team:Tsinghua/Human_Practices">Human practice</a></li> |
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<br> is detected by suvCas9, it can immediately trigger the suicidal system to eliminate the mutated | <br> is detected by suvCas9, it can immediately trigger the suicidal system to eliminate the mutated | ||
<br> cell, avoiding further catastrophic consequences. | <br> cell, avoiding further catastrophic consequences. | ||
+ | <br> | ||
+ | <br> | ||
+ | <div style="margin-left:170px;width:30%;height:30%"><img src="https://static.igem.org/mediawiki/2016/8/80/T--Tsinghua--suvCas9.PNG"></div> | ||
+ | <br><strong style="margin-left:130px"></strong>The composition of suvCas9 designed in our project | ||
<br> | <br> | ||
<h2 class="contentPage"><br><i>In vivo</i> generation of ssDNA by SCRIBE system</h2> | <h2 class="contentPage"><br><i>In vivo</i> generation of ssDNA by SCRIBE system</h2> | ||
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<br> and it avoids any unwanted targeting in the nucleus. However, the synthesis of such sequence is not | <br> and it avoids any unwanted targeting in the nucleus. However, the synthesis of such sequence is not | ||
<br> convenient, and the application in yeasts has not been tested before, not to mention such single-stranded | <br> convenient, and the application in yeasts has not been tested before, not to mention such single-stranded | ||
− | <br> nucleotides are hard to be generated and replicated <i>in vivo</i>. | + | <br> nucleotides are hard to be generated and replicated <i>in vivo</i>. Illustration is placed at the bottom |
+ | <br> of this page. | ||
<br> | <br> | ||
<br>The limitations of direct introduction of PAMmer sequence into yeast create a niche for us to find | <br>The limitations of direct introduction of PAMmer sequence into yeast create a niche for us to find | ||
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<br> cell on the counter-selection media and the fluorescence expression that can be quantitatively detected | <br> cell on the counter-selection media and the fluorescence expression that can be quantitatively detected | ||
<br>by FACS for further optimization of our surveillance system.<br><br> | <br>by FACS for further optimization of our surveillance system.<br><br> | ||
− | <div style="margin-left: | + | <div style="margin-left:150px;width:50%;height:50%"><img src="https://static.igem.org/mediawiki/2016/9/9e/T--Tsinghua--Mechanism1.PNG"></div> |
+ | <br> | ||
+ | <div style="margin-left:150px;width:50%;height:50%"><img src="https://static.igem.org/mediawiki/2016/c/c2/T--Tsinghua--Mechanism2.PNG"></div> | ||
+ | <br> | ||
+ | <div style="margin-left:150px;width:50%;height:50%"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tsinghua--Mechanism3.PNG"></div> | ||
+ | <br> | ||
+ | <div style="margin-left:150px;width:50%;height:50%"><img src="https://static.igem.org/mediawiki/2016/b/b5/T--Tsinghua--mechanism4.PNG"></div> | ||
<br>Figure 1A. A schematic overview of the targeting mechanism of suvCas9. suvCas9 can bind to a correct | <br>Figure 1A. A schematic overview of the targeting mechanism of suvCas9. suvCas9 can bind to a correct | ||
<br> mRNA in complex with sgRNA and PAMmer (illustrated above), which can then be translocated into the | <br> mRNA in complex with sgRNA and PAMmer (illustrated above), which can then be translocated into the | ||
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<br> | <br> | ||
<br> | <br> | ||
− | <div style="margin-left:250px"><img src="https://static.igem.org/mediawiki/2016/ | + | <div style="margin-left:250px"><img src="https://static.igem.org/mediawiki/2016/b/b2/T--Tsinghua--figure1A.png"></div> |
<br>Figure 1B. Mechanism of the generation of ssDNA <i>in vivo</i>. A single strand DNA can be derived | <br>Figure 1B. Mechanism of the generation of ssDNA <i>in vivo</i>. A single strand DNA can be derived | ||
<br> from the reversely transcribed product by the retron (RT). Figures are adapted from (7) and (8). | <br> from the reversely transcribed product by the retron (RT). Figures are adapted from (7) and (8). | ||
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<br> in living cells. Proc. Natl. Acad. Sci. USA 95, 11538–11543. | <br> in living cells. Proc. Natl. Acad. Sci. USA 95, 11538–11543. | ||
<br>[6] Fouts, D.E., True, H.L., and Celander, D.W. (1997). Functional recognition of fragmented operator sites | <br>[6] Fouts, D.E., True, H.L., and Celander, D.W. (1997). Functional recognition of fragmented operator sites | ||
− | <br> by R17/MS2 coat protein, a translational repressor. Nucleic Acids Res. 25, 4464–4473. | + | <br> by R17/MS2 coat protein, a translational repressor. Nucleic Acids Res. 25, 4464–4473.<br> |
<br>[7] Nelles, D. A., Fang, M. Y., O’Connell, M. R., Xu, J. L., Markmiller, S. J., Doudna, J. A., & Yeo, | <br>[7] Nelles, D. A., Fang, M. Y., O’Connell, M. R., Xu, J. L., Markmiller, S. J., Doudna, J. A., & Yeo, | ||
− | <br> G. W. (2016). Programmable RNA Tracking in Live Cells with CRISPR/Cas9. Cell, 165(2), 488-496. | + | <br> G. W. (2016). Programmable RNA Tracking in Live Cells with CRISPR/Cas9. Cell, 165(2), 488-496.<br> |
<br>[8] Farzadfard, F., & Lu, T. K. (2014). Genomically encoded analog memory with precise in vivo DNA writing | <br>[8] Farzadfard, F., & Lu, T. K. (2014). Genomically encoded analog memory with precise in vivo DNA writing | ||
<br> in living cell populations. Science, 346(6211), 1256272. | <br> in living cell populations. Science, 346(6211), 1256272. | ||
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<br>[10] Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., ... & Koonin, E. V. | <br>[10] Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., ... & Koonin, E. V. | ||
<br> (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186-191. | <br> (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186-191. | ||
− | <br>[11] Sherman, F. (1991). [1] Getting started with yeast. Methods in enzymology, 194, 3-21. | + | <br>[11] Sherman, F. (1991). [1] Getting started with yeast. Methods in enzymology, 194, 3-21.<br> |
<br>[12] O’Connell, M.R., Oakes, B.L., Sternberg, S.H., East-Seletsky, A., Kaplan, M., and Doudna, J.A. (2014). | <br>[12] O’Connell, M.R., Oakes, B.L., Sternberg, S.H., East-Seletsky, A., Kaplan, M., and Doudna, J.A. (2014). | ||
− | <br> Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266. | + | <br> Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266. <br> |
<br>[13] Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., & Boeke, J. D. (1996). Reverse two-hybrid and | <br>[13] Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., & Boeke, J. D. (1996). Reverse two-hybrid and | ||
<br> one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proceedings | <br> one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proceedings | ||
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