Difference between revisions of "Team:Fudan/Basic Parts"

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                         <span>Here we present our library of basic parts </span>
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                     <h1>Basic parts</h1>
 
                     <h1>Basic parts</h1>
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                     <p><span>Here we present our library of basic parts</span></p>
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                    <li>Basic parts</li>
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                        <h2>Best part- mouse Notch 1 core</h2>
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                    <li class="onThisPageNav"><a href="#section1">xxx</a></li>
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                        <p>Our favorite part for this year’s iGEM competition is mouse Notch 1 receptor’s core domain.
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                    <li><a href="https://2018.igem.org/Team:Fudan/Composite_Parts">Composite parts</a></li>
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                        </p><p>
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                    <li><a href="https://2018.igem.org/Team:Fudan/Optimization">Optimization</a></li>
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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 cleavts 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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                    <li><a href="https://2018.igem.org/Team:Fudan/Parts_Collection">Parts collection</a></li>
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                    <li><a href="https://2018.igem.org/Team:Fudan/Improve">Parts improvement</a></li>
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                    <li><a href="https://2018.igem.org/Team:Fudan/Measurement">Quantification</a></li>
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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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                            <p>
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                                Figure: Structure of the negative regulatory region of Notch 1 receptor, surface delivery, and optimization.
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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)
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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.
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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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                            <table>
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                                <tr>
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                                    <th>SynNotch</th>
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                                    <th>Classa)</th>
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                                    <th>ARFaSb)</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>
 +
                                    <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>
 +
                                    <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>
 +
                                    <td>2.12 ± 0.39</td>
 +
                                    <td>☆</td>
 +
                                </tr>
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                                <tr>
 +
                                    <td>LaG17-LNRAlnkr(L1457G)mN1c-tTAA</td>
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                                    <td>II</td>
 +
                                    <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>See more in <a href="https://2018.igem.org/Team:Fudan/Optimization">Optimization</a></p>
 +
                        </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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Revision as of 01:59, 18 October 2018

Basic parts

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Basic parts

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Best part- mouse Notch 1 core

Our favorite 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 cleavts 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.

Figure: 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 Classa) ARFaSb) 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

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.

Abstract

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.