Difference between revisions of "Team:SMMU-China/Part Collection"

Line 158: Line 158:
 
             <a style="text-decoration-color: HighlightText">(figure 1a)</a>. CAR-expressing NK cells can
 
             <a style="text-decoration-color: HighlightText">(figure 1a)</a>. CAR-expressing NK cells can
 
             detect tumor cells with HER2 on the surface and attack them.</p>
 
             detect tumor cells with HER2 on the surface and attack them.</p>
 +
        <p>We investigated the anti-tumor potential of the CAR-transduced NK-92 cells by standard 51Cr-release
 +
            assays. </p>
 +
        <img style="width: 85%;" src="https://static.igem.org/mediawiki/2019/c/c7/T--SMMU-China--partcollection_3.png"
 +
            alt="smmu_demonstrate_1" style="margin: 10px 0px;">
 +
        <center>Figure. 2 Results of two types of CARs.
 +
        </center>
 +
        <h2 style="text-align: left">SynNotch</h2>
 +
        <p>SynNotch receptors are synthetic receptors improved from native Notch. This kind of receptors are activated
 +
            by binding to surface-bound antigens on neighboring cells, which then trigger release of a synthetic
 +
            transcription factor to modulate target gene expression<sup>[1]</sup>. </p>
 +
        <p>Our synthetic receptor synNotch contained an extracellular scFv against antigen HER2 <a
 +
                href="http://parts.igem.org/Part:BBa_K3132005" style="text-decoration: underline">(BBa_K3132005)</a>
 +
            bound on
 +
            the surface of tumor cells, a transmembrane NL domain through cytoplasmic <a
 +
                    href="http://parts.igem.org/Part:BBa_K3132005" style="text-decoration: underline">(BBa_K3132005)</a>
 +
            and an
 +
            intracellular domain which was set as synthetic transcription factor.<a
 +
                    style="text-decoration-color: HighlightText">(figure 1b)</a>.</p>
 +
        <p>To characterize synNotch, we firstly found two breast cancer cell lines expressing high and low level of HER2
 +
            respectively by flow cytometry. Then we tested the response of synNotch with three different transcription
 +
            factors fused to intracellular segments. The results show that synNotch had high sensitivity to HER2
 +
            signals, moreover, we can optimize its function by fusing the most suitable transcription factors to the
 +
            intracellular domain.<a
 +
                    style="text-decoration-color: HighlightText">(Figure 3)</a></p>
 +
        <img style="width: 50%;" src="https://static.igem.org/mediawiki/2019/3/34/T--SMMU-China--partcollection_4.png"
 +
            alt="smmu_demonstrate_1" style="margin: 10px 0px;">
 +
        <center>Figure 3. Response of three types of synNotch with different transcription factors Gal4-VP64, PIP-VP64
 +
            and ZF21-16-VP64.
 +
        </center>
 +
        <h1 style="text-align: left">Receptors for soluble molecules:</h1>
 +
        <h2 style="text-align: left">MESA</h2>
 +
        <p>Modular extracellular sensor architecture, also known as MESA, is also a kind of designable synthetic
 +
            receptors. This kind of receptors are made up of two different chains, PC chain and TC chain. After being
 +
            induced by ligands, the two chains will dimer and interact with each other. One chain is fused to the TF by
 +
            a protease cleavage site and the other chain contains a TEV protease<sup>[1]</sup>. After ligand-induced
 +
            dimerization,
 +
            the protease will recognize the cleavage site and cut down the TF to regulate gene expression.</p>
 +
        <p>Our synthetic receptors MESA contained an extracellular scFv against SunTag <a
 +
                href="http://parts.igem.org/Part:BBa_K3132007" style="text-decoration: underline">(BBa_K3132007)</a>
 +
            released by core
 +
            device, a transmembrane domain based on a submitted parts <a
 +
                    href="http://parts.igem.org/Part:BBa_K1065102" style="text-decoration: underline">(BBa_K1065102)</a>
 +
            and an intracellular domain with
 +
            transcription factor Gal4-VP64 connected to the protease cleavage site.<a
 +
                    style="text-decoration-color: HighlightText">(figure 1c)</a>.</p>
 +
        <img style="width: 50%;" src="https://static.igem.org/mediawiki/2019/e/e3/T--SMMU-China--partcollection_5.png"
 +
            alt="smmu_demonstrate_1" style="margin: 10px 0px;">
 +
        <center>Figure. 4 Response of three types of MESA: anti-SunTag PC+anti-SunTag TC (S+S), anti-SunTag PC+anti-IL
 +
            TC (S+I), anti-IL PC+anti-SunTag TC (I+S). S+I MESA showed a significant response to 6xSunTag-IL ligand.
 +
        </center>
 +
        <h2 style="text-align: left">GEMS</h2>
 +
        <p>Generalized extracellular molecule sensor, also known as GEMS, is generalizable because it could be adapted
 +
            to sense a wide range of input signals and express genes as the users wish<sup>[1]</sup>. Like MESA, GEMS
 +
            contains two
 +
            functional chains, however, it exists in the form of dimerization. Ligand binding to the receptors is
 +
            thought to rotate each chain subunit around its own axis then the activated receptor can induce downstream
 +
            cascade. </p>
 +
        <p>Our synthetic receptor GEMS contained an extracellular scFv against suntag <a
 +
                href="http://parts.igem.org/Part:BBa_K3132018" style="text-decoration: underline">((BBa_K3132018)</a>
 +
            released by core
 +
            device, a transmembrane scaffold based on the erythropoietin receptor and an intracellular domain fused to
 +
            FGFR1, which is able to activate MAPK pathway and regulate gene expression relied on transcription factors
 +
            PIP-Elk <a
 +
                    href="http://parts.igem.org/Part:BBa_K3132012"
 +
                    style="text-decoration: underline">((BBa_K3132012)</a><a
 +
                    style="text-decoration-color: HighlightText">(figure 1d)</a>.</p>
 +
        <p>We did large amount of experience to characterize GEMS and got some satisfactory results. We constructed
 +
            different lengths of suntags and tested the receptor's response to these labeled proteins. The results show
 +
            that GEMS had the most sensitivity to 6xsuntag, and well activation of the target gene.<a
 +
                    style="text-decoration-color: HighlightText">(figure5)</a></p>
 +
        <img style="width: 90%;" src="https://static.igem.org/mediawiki/2019/2/20/T--SMMU-China--partcollection_6.png"
 +
            alt="smmu_demonstrate_1" style="margin: 10px 0px;">
 +
        <center>Figure 5. Response of GEMS to three ligands. a, Compared with 2x and 4xSunTag proteins, GEMS only
 +
            responded to the 6x ligand in a concentration-dependent way. b, mCherry expressed by cells at different
 +
            6xSunTag concentrations.
 +
        </center>
 +
        <p>Here we collected these four types of synthetic receptors, hoping to construct a toolbox for creating devices
 +
            that sense and respond to user-defined ligands, capitalizing on native ligand-receptor interactions and
 +
            ultimately rewiring cascades and regulating user-defined responses. More information can be searched on
 +
            their pages.</p>
 +
        <div align="center">
 +
            <h2>Receptors for soluble molecules</h2>
 +
            <table class="smmu-table">
 +
                <thead>
 +
                <tr>
 +
                    <th>GEMS</th>
 +
                    <th>MESA</th>
 +
                </tr>
 +
                </thead>
 +
                <tbody>
 +
                <tr>
 +
                    <td data-label="GEMS"><a href="http://parts.igem.org/Part:BBa_K3132016"
 +
                                                                target="_blank">(BBa_K3132016)</a>
 +
                    </td>
 +
                    <td data-label="MESA"><a href="http://parts.igem.org/Part:BBa_K1065102"
 +
                                                                target="_blank">(BBa_K1065102)</a>
 +
                    </td>
 +
                </tr>
 +
                </tbody>
 +
            </table>
 +
        </div>
 +
        <div align="center">
 +
            <h2>Receptors for surface-bound molecules</h2>
 +
            <table class="smmu-table">
 +
                <thead>
 +
                <tr>
 +
                    <th>CAR</th>
 +
                    <th>synNotch</th>
 +
                </tr>
 +
                </thead>
 +
                <tbody>
 +
                <tr>
 +
                    <td data-label="GEMS"><a href="http://parts.igem.org/Part:BBa_K3132015"
 +
                                            target="_blank">(BBa_K3132015)</a>
 +
                    </td>
 +
                    <td data-label="MESA"><a href="http://parts.igem.org/Part:BBa_K3132020"
 +
                                            target="_blank">(BBa_K3132020)</a>
 +
                    </td>
 +
                </tr>
 +
                </tbody>
 +
            </table>
 +
        </div>
 
     </div>
 
     </div>
  

Revision as of 12:41, 21 October 2019

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Part Collection
This is a descriptive paragraph.

1.Synthetic receptors

OverView

Transmembrane receptors can continuously monitor the extracellular milieu, and transduce specific extracellular signals into intracellular responses. Binding of ligands that match the receptors triggers intracellular signaling cascades and ultimately induces transcriptional changes[1]. We found some latest receptors, with some of them sensing and responding to soluble molecules while others sensing and responding to surface-bound molecules. We placed them in our different devices, giving them different roles, and compared their function to determine the most suitable one.

smmu_demonstrate_1 smmu_demonstrate_1
Figure 1. The structure and mechanism of synthetic receptors we chose. a. chimeric antigen receptor (CAR) b. SynNotch c. Modular extracellular sensor architecture (MESA) d. Generalized extracellular molecule sensor (GEMS)

Receptors for surface-bound molecules:

CAR

The best-known examples of synthetic receptors that sense and respond to molecules bound to the surface of other cells are the so-called chimeric antigen receptors (CARs). Based on large number of researches and characterization of CARs, we chose it as the core tool to achieve the killing function of the core device.

Our synthetic receptor CARs contained an extracellular scFv trastuzumab (BBa_K3132022) and an intracellular regulatory domain of the NK-cell receptor complex (BBa_K3132021) (figure 1a). CAR-expressing NK cells can detect tumor cells with HER2 on the surface and attack them.

We investigated the anti-tumor potential of the CAR-transduced NK-92 cells by standard 51Cr-release assays.

smmu_demonstrate_1
Figure. 2 Results of two types of CARs.

SynNotch

SynNotch receptors are synthetic receptors improved from native Notch. This kind of receptors are activated by binding to surface-bound antigens on neighboring cells, which then trigger release of a synthetic transcription factor to modulate target gene expression[1].

Our synthetic receptor synNotch contained an extracellular scFv against antigen HER2 (BBa_K3132005) bound on the surface of tumor cells, a transmembrane NL domain through cytoplasmic (BBa_K3132005) and an intracellular domain which was set as synthetic transcription factor.(figure 1b).

To characterize synNotch, we firstly found two breast cancer cell lines expressing high and low level of HER2 respectively by flow cytometry. Then we tested the response of synNotch with three different transcription factors fused to intracellular segments. The results show that synNotch had high sensitivity to HER2 signals, moreover, we can optimize its function by fusing the most suitable transcription factors to the intracellular domain.(Figure 3)

smmu_demonstrate_1
Figure 3. Response of three types of synNotch with different transcription factors Gal4-VP64, PIP-VP64 and ZF21-16-VP64.

Receptors for soluble molecules:

MESA

Modular extracellular sensor architecture, also known as MESA, is also a kind of designable synthetic receptors. This kind of receptors are made up of two different chains, PC chain and TC chain. After being induced by ligands, the two chains will dimer and interact with each other. One chain is fused to the TF by a protease cleavage site and the other chain contains a TEV protease[1]. After ligand-induced dimerization, the protease will recognize the cleavage site and cut down the TF to regulate gene expression.

Our synthetic receptors MESA contained an extracellular scFv against SunTag (BBa_K3132007) released by core device, a transmembrane domain based on a submitted parts (BBa_K1065102) and an intracellular domain with transcription factor Gal4-VP64 connected to the protease cleavage site.(figure 1c).

smmu_demonstrate_1
Figure. 4 Response of three types of MESA: anti-SunTag PC+anti-SunTag TC (S+S), anti-SunTag PC+anti-IL TC (S+I), anti-IL PC+anti-SunTag TC (I+S). S+I MESA showed a significant response to 6xSunTag-IL ligand.

GEMS

Generalized extracellular molecule sensor, also known as GEMS, is generalizable because it could be adapted to sense a wide range of input signals and express genes as the users wish[1]. Like MESA, GEMS contains two functional chains, however, it exists in the form of dimerization. Ligand binding to the receptors is thought to rotate each chain subunit around its own axis then the activated receptor can induce downstream cascade.

Our synthetic receptor GEMS contained an extracellular scFv against suntag ((BBa_K3132018) released by core device, a transmembrane scaffold based on the erythropoietin receptor and an intracellular domain fused to FGFR1, which is able to activate MAPK pathway and regulate gene expression relied on transcription factors PIP-Elk ((BBa_K3132012)(figure 1d).

We did large amount of experience to characterize GEMS and got some satisfactory results. We constructed different lengths of suntags and tested the receptor's response to these labeled proteins. The results show that GEMS had the most sensitivity to 6xsuntag, and well activation of the target gene.(figure5)

smmu_demonstrate_1
Figure 5. Response of GEMS to three ligands. a, Compared with 2x and 4xSunTag proteins, GEMS only responded to the 6x ligand in a concentration-dependent way. b, mCherry expressed by cells at different 6xSunTag concentrations.

Here we collected these four types of synthetic receptors, hoping to construct a toolbox for creating devices that sense and respond to user-defined ligands, capitalizing on native ligand-receptor interactions and ultimately rewiring cascades and regulating user-defined responses. More information can be searched on their pages.

Receptors for soluble molecules

GEMS MESA
(BBa_K3132016) (BBa_K1065102)

Receptors for surface-bound molecules

CAR synNotch
(BBa_K3132015) (BBa_K3132020)

DEMO PART 2

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DEMO PART 3

Your team has been approved and you are ready to start the iGEM season! Your team has been approved and you are ready to start the iGEM season! Your team has been approved and you are ready to start the iGEM season! Your team has been approved and you are ready to start the iGEM season! Your team has been approved and you are ready to start the iGEM season! Your team has been approved and you are ready to start the iGEM season! Your team has been approved and you are ready to start the iGEM season!

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