Difference between revisions of "Team:Fudan-TSI/Basic Part"

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This html document is created by Tian Huang for Team Fudan iGEM 2018.
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2019.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2019.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2019.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>Basic Parts</h1>
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A <b>basic part</b> is a functional unit of DNA that cannot be subdivided into smaller component parts. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0051">BBa_R0051</a> is an example of a basic part, a promoter regulated by lambda cl.
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are <b>many</b> opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
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<h3>Note</h3>
 
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<p>This page should list all the basic parts your team has made during your project and include direct links to your Parts main pages on the Registry. <b>You must add all characterization information for your parts on Parts Main Page on the Registry.</b> You should <b>not</b> put characterization information on this page. Remember judges will only look at the first part in the list for the Best Basic Part award, so put your best part first!</p>
 
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<h3>Best Basic Part Special Prize</h3>
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    <title>2019 Team:Fudan -Basic_Part</title>
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<p> To be eligible for this award, this part <b>must be well documented on the part's Main Page on the Registry</b>. If you have a part you wish to nominate your team for this <a href="https://2019.igem.org/Judging/Awards">special prize</a>, make sure you add your part number to your <a href="https://2019.igem.org/Judging/Judging_Form">judging form</a> and delete the alert box at the top of this page.
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<b>Please note:</b> Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. </p>
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                        <h1>Basic parts</h1>
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                        <span>&nbsp;</span>
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                    <h1>Basic parts</h1>
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                    <p><span>&nbsp;</span></p>
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                    <div id="section1" class="section container">
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                        <h2>BBa_K2549006 <a href="http://parts.igem.org/Part:BBa_K2549006" target=_blank>mouse Notch 1 core</a></h2>
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                        <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" />
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                        </p><p>
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                            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.
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                        </p>
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                        <div class="expFigureHolder" style="width:100%">
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                                <div class="col s12 m4"><img style="width:100%" src="https://static.igem.org/mediawiki/2018/9/9e/T--Fudan--bestpart1.png"></div>
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                                <div class="col s12 m4"><img style="width:100%" src="https://static.igem.org/mediawiki/2018/f/f1/T--Fudan--bestpart2.png"></div>
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                                <div class="col s12 m4"><img style="width:100%" src="https://static.igem.org/mediawiki/2018/8/80/T--Fudan--opt-gif.gif"></div>
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                                <div class="col s12 m4 offset-m1"><img style="width:100%" src="https://static.igem.org/mediawiki/2018/2/21/T--Fudan--bestpart3.png"></div>
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                                <div class="col s12 m4 offset-m2"><img style="width:100%" src="https://static.igem.org/mediawiki/2018/8/81/T--Fudan--bestpart4.png"></div>
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                            </div>
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                            <p><b>Structure of the negative regulatory region of Notch 1 receptor, surface delivery, and optimization.</b><br/>
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                                        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/>
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                                        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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                                        e. A conclusive diagram of our Notch optimization. We have 5 mutations that exhibit better activation performance to SynNotch with LaG17 as scFv.
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                            </p>
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                        </div>
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                        <b class="tableHolder">
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                            <table>
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                                <tr>
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                                    <th>SynNotch</th>
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                                    <th>Class</th>
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                                    <th>ARF<sub>aS</sub><sup>*)</sup></th>
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                                    <th>Preferred</th>
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                                </tr>
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                                <tr>
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                                    <td>LaG17-mN1c-tTAA</td>
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                                    <td>II</td>
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                                    <td>1.67 ± 0.20</td>
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                                    <td></td>
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                                </tr>
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                                <tr>
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                                    <td>LaG17-6G-LNRA[-]mN1c-tTAA</td>
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                                    <td>I</td>
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                                    <td>1.73 ± 0.29</td>
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                                    <td>☆</td>
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                                </tr>
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                                <tr>
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                                    <td>LaG17-LNRA cbs(D1433K)mN1c-tTAA</td>
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                                    <td>II</td>
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                                    <td>2.69 ± 0.11</td>
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                                    <td>☆</td>
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                                </tr>
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                                <tr>
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                                    <td>LaG17-LNRA cbs(D1433Q)mN1c-tTAA</td>
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                                    <td>II</td>
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                                    <td>Needs more repeat</td>
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                                    <td></td>
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                                </tr>
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                                <tr>
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                                    <td>LaG17-LNRAlnkr(L1457V)mN1c-tTAA</td>
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                                    <td>I</td>
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                                    <td>2.12 ± 0.39</td>
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                                    <td>☆</td>
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                                </tr>
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                                <tr>
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                                    <td>LaG17-LNRAlnkr(L1457G)mN1c-tTAA</td>
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                                    <td>II</td>
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                                    <td>2.55± 0.55</td>
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                                    <td></td>
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                                </tr>
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                            </table>
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                            <p>*) <b>ARF<sub>aS</sub>:</b> activation ratio fold of activating-form SynNotch</p>
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                            <p>See more in <a href="/Team:Fudan-TSI/Optimization">Optimization</a></p>
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                        </div>
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                        <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.
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                        </p>
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                    </div>
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                </main>
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            </div>
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            <!--Abstract on content page-->
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            <div id="abstractContent" class="z-depth-2">
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                <a href="#!"><img alt=alt="project summary" src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a>
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                <div class="container">
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                    <h2 style="margin: 0;padding: 10px 0;">Project Summary</h2>
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                    <p style="margin: 0">Contact-dependent signaling is critical for multicellular biological
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                        events, yet customizing contact-dependent signal transduction between
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                        cells remains challenging. Here we have developed the ENABLE toolbox, a
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                        complete set of transmembrane binary logic gates. Each gate consists of
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                        3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic
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                        Notch receptors to enable cells to respond to different signals across the
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                        membrane reliably. These signals, individually amplified intracellularly by
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                        transcription, are further combined for computing. Our engineered zinc finger-based
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                        transcription factors perform binary computation and output designed products.
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                        In summary, we have combined spatially different signals in mammalian cells,
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                        and revealed new potentials for biological oscillators, tissue engineering,
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                        cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing
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                        contact-dependent signaling networks in mammals. The 3-layer design principle
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                        underlying ENABLE empowers any future development of transmembrane logic circuits,
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                        thus contributes a foundational advance to Synthetic Biology.
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                    </p>
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                            </a><a href="http://www.fudan.edu.cn/en/" target="_blank"><img class="col s3 m6 l3" alt="Fudan University" src="https://static.igem.org/mediawiki/2018/f/f7/T--Fudan--schoolLogo.png">
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Revision as of 06:25, 29 August 2019

<script src="https://ajax.aspnetcdn.com/ajax/jQuery/jquery-1.11.3.min.js"></script> 2019 Team:Fudan -Basic_Part

Basic parts

 

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.
006 screencapture

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.

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Project Summary

Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.