Difference between revisions of "Team:Fudan-TSI/Basic Part"
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<title>2019 Team:Fudan-TSI Basic_Part</title> | <title>2019 Team:Fudan-TSI Basic_Part</title> | ||
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| − | <p style="width: 100%;text-align: center | + | <p class="flow-text" style="width:100%;text-align:center"><span class="white-text">Basic parts</span></p> |
</div></li> | </div></li> | ||
<li> | <li> | ||
<ul class="collapsible expandable"> | <ul class="collapsible expandable"> | ||
| − | <li>Team: Fudan-TSI</li> | + | <li class="onThisPageNav"><span>Team: Fudan-TSI</span></li> |
| − | <li><div class="collapsible-header">Project</div> | + | <li><div class="collapsible-header"><span>Project</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Description">Background</a></li> | <li><a href="/Team:Fudan-TSI/Description">Background</a></li> | ||
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</ul></div> | </ul></div> | ||
</li> | </li> | ||
| − | <li><div class="collapsible-header">Results</div> | + | <li><div class="collapsible-header"><span>Results</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Results#ReverseTranscription">Reverse Transcription</a></li> | <li><a href="/Team:Fudan-TSI/Results#ReverseTranscription">Reverse Transcription</a></li> | ||
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| − | <li><div class="collapsible-header">Model</div> | + | <li><div class="collapsible-header"><span>Model</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Model">Modeling</a></li> | <li><a href="/Team:Fudan-TSI/Model">Modeling</a></li> | ||
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| − | <li><div class="collapsible-header">Parts</div> | + | <li><div class="collapsible-header"><span>Parts</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Basic_Part">Basic parts</a></li> | <li><a href="/Team:Fudan-TSI/Basic_Part">Basic parts</a></li> | ||
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| − | <li><div class="collapsible-header">Outreach</div> | + | <li><div class="collapsible-header"><span>Outreach</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Public_Engagement">Education & Public engagement</a></li> | <li><a href="/Team:Fudan-TSI/Public_Engagement">Education & Public engagement</a></li> | ||
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| − | <li><div class="collapsible-header">Team</div> | + | <li><div class="collapsible-header"><span>Team</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Team">Members</a></li> | <li><a href="/Team:Fudan-TSI/Team">Members</a></li> | ||
<li><a href="/Team:Fudan-TSI/Attributions">Attributions</a></li> | <li><a href="/Team:Fudan-TSI/Attributions">Attributions</a></li> | ||
<li><a href="https://2018.igem.org/Team:Fudan/Heritage" target=_blank>Heritage</a></li> | <li><a href="https://2018.igem.org/Team:Fudan/Heritage" target=_blank>Heritage</a></li> | ||
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<h1>Basic parts</h1> | <h1>Basic parts</h1> | ||
| − | <p><span> </span></p> | + | <p class="flow-text"><span> </span></p> |
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<h2>BBa_K2549006 <a href="http://parts.igem.org/Part:BBa_K2549006" target=_blank>mouse Notch 1 core</a></h2> | <h2>BBa_K2549006 <a href="http://parts.igem.org/Part:BBa_K2549006" target=_blank>mouse Notch 1 core</a></h2> | ||
| − | <p>Our favorite basic part for this year’s iGEM competition is mouse Notch 1 receptor’s core domain.<br/><img alt="006 screencapture" src="https://static.igem.org/mediawiki/2018/thumb/7/7e/T--Fudan--BBa_K2549006.png/1444px-T--Fudan--BBa_K2549006.png" /> | + | <p class="flow-text">Our favorite basic part for this year’s iGEM competition is mouse Notch 1 receptor’s core domain.<br/><img alt="006 screencapture" src="https://static.igem.org/mediawiki/2018/thumb/7/7e/T--Fudan--BBa_K2549006.png/1444px-T--Fudan--BBa_K2549006.png" /> |
| − | </p><p> | + | </p><p class="flow-text"> |
Notch core consists of the negatively regulated region and transmembrane region of the mouse Notch 1 receptor. It is the joint that empowers SynNotch the high modularity and proper activation. From the N terminal to C terminal, it is composed of three lin12-repeats (LNR) domains LNR-A, LNR-B, LNR-C, the heterodimerization domain (HD), and the transmembrane domain. The LNR-AB linker between LNR-A and B is like a plug that occludes ADAM from cleaving wild type extracellular domain can be substituted by antiGFP scFvs like LaG16 and anti-CD19, while its intracellular domain can be replaced by specialized transcription factors. Notch core is also the key to the activation of both wild type Notch receptors and SynNotch. | Notch core consists of the negatively regulated region and transmembrane region of the mouse Notch 1 receptor. It is the joint that empowers SynNotch the high modularity and proper activation. From the N terminal to C terminal, it is composed of three lin12-repeats (LNR) domains LNR-A, LNR-B, LNR-C, the heterodimerization domain (HD), and the transmembrane domain. The LNR-AB linker between LNR-A and B is like a plug that occludes ADAM from cleaving wild type extracellular domain can be substituted by antiGFP scFvs like LaG16 and anti-CD19, while its intracellular domain can be replaced by specialized transcription factors. Notch core is also the key to the activation of both wild type Notch receptors and SynNotch. | ||
</p> | </p> | ||
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| − | <p><b>Structure of the negative regulatory region of Notch 1 receptor, surface delivery, and optimization.</b><br/> | + | <p class="flow-text"><b>Structure of the negative regulatory region of Notch 1 receptor, surface delivery, and optimization.</b><br/> |
a-c. Shows structure of the Notch 1 core, with more detail on the NRR. (With structural information adapted from Gordon et al., 2009)<br/> | a-c. Shows structure of the Notch 1 core, with more detail on the NRR. (With structural information adapted from Gordon et al., 2009)<br/> | ||
d. Shows that mouse SynNotch with LaG16-2 scFv (single chain fragment variants) as ectodomain is expressed on the 293T cell membrane as an example via anti-myc AF488 immunofluorescence staining. Shown here the light parts.<br/> | d. Shows that mouse SynNotch with LaG16-2 scFv (single chain fragment variants) as ectodomain is expressed on the 293T cell membrane as an example via anti-myc AF488 immunofluorescence staining. Shown here the light parts.<br/> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
| − | <p>*) <b>ARF<sub>aS</sub>:</b> activation ratio fold of activating-form SynNotch</p> | + | <p class="flow-text">*) <b>ARF<sub>aS</sub>:</b> activation ratio fold of activating-form SynNotch</p> |
| − | <p>See more in <a href="/Team:Fudan-TSI/Optimization">Optimization</a></p> | + | <p class="flow-text">See more in <a href="/Team:Fudan-TSI/Optimization">Optimization</a></p> |
</div> | </div> | ||
| − | <p>It is suggested by recent research that Notch activation begin as the receptor is first "opened up" at its negatively regulatory region (NRR) by the mechanical force exerted by Notch-bound ligand endocytosis on signal-sender cell, exposing its cleavage sites to proteases. Intracellular proteolytic action releases the Notch intracellular domain (NICD). In the majority of cellular interactions, the free Notch intracellular domain then translocate into the cell nucleus via nuclear localization sequence to regulate downstream signaling. For wild type Notch, the Notch intracellular domain would interacted with its major downstream effector CBF-1/Suppressor of Hairless/Lag-1 (CSL) on their target DNA. Together they recruit co-factors to activate endogenous downstream transcription. For SynNotch, specialized transcription factors will exclusively participate in regulation of genetic circuits that allow user-defined cellular responses. | + | <p class="flow-text">It is suggested by recent research that Notch activation begin as the receptor is first "opened up" at its negatively regulatory region (NRR) by the mechanical force exerted by Notch-bound ligand endocytosis on signal-sender cell, exposing its cleavage sites to proteases. Intracellular proteolytic action releases the Notch intracellular domain (NICD). In the majority of cellular interactions, the free Notch intracellular domain then translocate into the cell nucleus via nuclear localization sequence to regulate downstream signaling. For wild type Notch, the Notch intracellular domain would interacted with its major downstream effector CBF-1/Suppressor of Hairless/Lag-1 (CSL) on their target DNA. Together they recruit co-factors to activate endogenous downstream transcription. For SynNotch, specialized transcription factors will exclusively participate in regulation of genetic circuits that allow user-defined cellular responses. |
</p> | </p> | ||
</div> | </div> | ||
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<a href="#!"><img alt="project summary" src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a> | <a href="#!"><img alt="project summary" src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a> | ||
<div class="container"> | <div class="container"> | ||
| − | < | + | <h4 style="margin:0;padding:10px 0;">Project Summary</h4> |
| − | <p style="margin: 0">Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for <i>in vivo</i> continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the <i>in vivo</i> evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology. | + | <p class="flow-text" style="margin:0">Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for <i>in vivo</i> continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the <i>in vivo</i> evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology. |
</p> | </p> | ||
</div> | </div> | ||
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</a><a href="http://www.yfc.cn/en/" target="_blank"><img class="col s3 m6 l3" style="padding: 0.15rem 0.9rem;" alt="Yunfeng Capital" src="https://static.igem.org/mediawiki/2018/e/e2/T--Fudan--yunfengLogo.png"> | </a><a href="http://www.yfc.cn/en/" target="_blank"><img class="col s3 m6 l3" style="padding: 0.15rem 0.9rem;" alt="Yunfeng Capital" src="https://static.igem.org/mediawiki/2018/e/e2/T--Fudan--yunfengLogo.png"> | ||
</a> | </a> | ||
| − | <h3 class="col s12" style="text-align: left; color: rgba(255, 255, 255, 0.8); font-size: | + | <h3 class="col s12" style="text-align: left; color: rgba(255, 255, 255, 0.8); font-size:12.5px">R-Evolution: an <i>in vivo</i> sequence-specific toolbox for continuous mutagenesis</h3> |
</div> | </div> | ||
<div id="usefulLinks" class="col m9 s12 row"> | <div id="usefulLinks" class="col m9 s12 row"> | ||
Revision as of 07:04, 17 September 2019
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Basic parts
BBa_K2549006 mouse Notch 1 core
Our favorite basic part for this year’s iGEM competition is mouse Notch 1 receptor’s core domain.
Notch core consists of the negatively regulated region and transmembrane region of the mouse Notch 1 receptor. It is the joint that empowers SynNotch the high modularity and proper activation. From the N terminal to C terminal, it is composed of three lin12-repeats (LNR) domains LNR-A, LNR-B, LNR-C, the heterodimerization domain (HD), and the transmembrane domain. The LNR-AB linker between LNR-A and B is like a plug that occludes ADAM from cleaving wild type extracellular domain can be substituted by antiGFP scFvs like LaG16 and anti-CD19, while its intracellular domain can be replaced by specialized transcription factors. Notch core is also the key to the activation of both wild type Notch receptors and SynNotch.
Structure of the negative regulatory region of Notch 1 receptor, surface delivery, and optimization.
a-c. Shows structure of the Notch 1 core, with more detail on the NRR. (With structural information adapted from Gordon et al., 2009)
d. Shows that mouse SynNotch with LaG16-2 scFv (single chain fragment variants) as ectodomain is expressed on the 293T cell membrane as an example via anti-myc AF488 immunofluorescence staining. Shown here the light parts.
e. A conclusive diagram of our Notch optimization. We have 5 mutations that exhibit better activation performance to SynNotch with LaG17 as scFv.
| SynNotch | Class | ARFaS*) | Preferred |
|---|---|---|---|
| LaG17-mN1c-tTAA | II | 1.67 ± 0.20 | |
| LaG17-6G-LNRA[-]mN1c-tTAA | I | 1.73 ± 0.29 | ☆ |
| LaG17-LNRA cbs(D1433K)mN1c-tTAA | II | 2.69 ± 0.11 | ☆ |
| LaG17-LNRA cbs(D1433Q)mN1c-tTAA | II | Needs more repeat | |
| LaG17-LNRAlnkr(L1457V)mN1c-tTAA | I | 2.12 ± 0.39 | ☆ |
| LaG17-LNRAlnkr(L1457G)mN1c-tTAA | II | 2.55± 0.55 |
*) ARFaS: activation ratio fold of activating-form SynNotch
See more in Optimization
It is suggested by recent research that Notch activation begin as the receptor is first "opened up" at its negatively regulatory region (NRR) by the mechanical force exerted by Notch-bound ligand endocytosis on signal-sender cell, exposing its cleavage sites to proteases. Intracellular proteolytic action releases the Notch intracellular domain (NICD). In the majority of cellular interactions, the free Notch intracellular domain then translocate into the cell nucleus via nuclear localization sequence to regulate downstream signaling. For wild type Notch, the Notch intracellular domain would interacted with its major downstream effector CBF-1/Suppressor of Hairless/Lag-1 (CSL) on their target DNA. Together they recruit co-factors to activate endogenous downstream transcription. For SynNotch, specialized transcription factors will exclusively participate in regulation of genetic circuits that allow user-defined cellular responses.
Project Summary
Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for in vivo continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the in vivo evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology.