Difference between revisions of "Team:CSU CHINA/Part Collection"

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{{CSU_CHINA}}
 
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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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    <div class="container-fluid Topbox" id="Top">
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            <h1 class="display-3">Improved Part</h1>
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    </div>
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    <div class="container">
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            <div class="row">
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                    <div id="col-md-8 offset-md-2">
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                            <div id="mRNA" style="width: 100%; margin-top:30px;margin-bottom: 30px;">
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                                    <div class="card">
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                                        <div class="card-header">
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                                            <a class="card-link" data-toggle="collapse" href="#mRNA1" style="color: black">Composite Part</a>
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                                        </div>
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                                        <div id="mRNA1" class="collapse show" data-parent="#mRNA">
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                                            <div class="card-body">
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                                                <figure>
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                        <table class="table table-hover">
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                            <thead class="thead-inverse">
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                                <tr>
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                                        <td>Section</td>
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                                        <td>Name</td>
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                                        <td>Type</td>
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                                        <td>Description</td>
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                                </tr>
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                                </thead>
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                                <tbody>
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                                    <tr>
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                                        <td>New part</td>
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                                        <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2908676">BBa_K2908676</a></td>
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                                        <td>DNA</td>
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                                        <td>s(GATA3)p-GAD-miR101-BS</td>
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                                    </tr>
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                                    <tr>
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                                        <td>Existing part</td>
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                                        <td><a href="http://parts.igem.org/Part:BBa_K2580666">BBa_K2580666</td>
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                                        <td>DNA</td>
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                                        <td>CMV-Gal4-VP16</td>
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                                    </tr>
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                                </tbody>
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                        </table>
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                    </div>
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                </center>
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            </figure>
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        </div>
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    </div>
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                        <h3>New part</h3>
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                        <img src="https://static.igem.org/mediawiki/2019/7/77/T--CSU_CHINA--Improved_part.png" class="mx-auto" style="width: 80% ">
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                        <p class="ts"><b>s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]</b></p>
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<div class="column full_size">
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                        <div class="d-flex justify-content-center">
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                                <p style="color: grey; width: 80%; margin-bottom: 32px;text-align: center;">The part improved in the specificity in Triple negative cancer (TNBC). </p>
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<h1> Part Collection </h1>
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                        </div>
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<p>Did your team make a lot of great parts? Is there a theme that ties all your parts together? Do you have more than 10 parts in this collection? Did you make a CRISPR collection, a MoClo collection, or a collection of awesome pigment parts? Describe your parts collection on this page, so the judges can evaluate you for the Best Part Collection award.</p>
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                        <br>
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                        <h3>Existing part</h3>
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<p>
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                        <img src="https://static.igem.org/mediawiki/2019/0/00/T--CSU_CHINA--CMV-Gal4-VP16.png" class="mx-auto" style="width: 80% ">
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While you should put all the characterization information for your parts on the Registry, you are encouraged to explain how all your parts form a collection on this page.  
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                        <p class="ts"><b>CMV-Gal4-VP16 (BBa_K2580666)</b></p>
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</p>
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                        <div class="d-flex justify-content-center">
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</div>
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                                <p style="color: grey; width: 80%; margin-bottom: 32px;text-align: center;">The part has low specificity in the TNBC cells compared to normal cells although the team members who created the part stated that it is a cancer-specific part.</p>
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                        </div>
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                        <p>As for this new part we created this year, it contains the brand new synthetic promoter we designed (BBa_K2908000),  the existing sequence in the previous iGEM parts, GAD (Gal4-VP16), and the new designed part miR101-BD. </p>
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<div class="column full_size">
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                        <p>In our project, we wanted to make the expression of GAD to be specific in Triple negative breast cancer cells (TNBC cells). After communicating with the team creating the part who says that in breast cancer, this part has higher efficiency in cancer cells than normal cells, we tested the efficiency of GAD expression by co-transfecting the CMV-Gal4-VP16 plasmid and pGL4.22-G8p (pGL4.22 has a luciferase gene after G8p, a promoter can be driven by Gal4-VP16) into TNBC cells (cancer cells) and normal cells. A in the figure below shows the results.</p>
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<div class="highlight decoration_background">
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                        <p>Then, we <strong>exchange the promoter CMV into s(GATA3)p, and added a miR101-BD in the 3’UTR of the gene GAD (Gal4-VP16)</strong>, as miR101 is low expressed in the cancer cells while high in the normal cells [1] (the binding of miR101 to miR101-BD can inhibit the expression of GAD protein, then to down-regulate the efficiency of G8p). We used the same method to test the efficiency of our part, that is, co-transfecting the s(GATA3)p-GAD-miR101-BD plasmid and pGL4.22-G8p, and doing the luciferase assay [2]. The figure B shows the results. We also compared the specificity between CMV-Gal4-VP16 and s(GATA3)p-GAD-miR101-BD (C), which indicated that our part dramatically improved the function in specifically regulating the expression of GAD.</p>
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<h3>Note</h3>
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                        <p></p>
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<p>This page should list all the parts in the collection your team 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.
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                        <img src="https://static.igem.org/mediawiki/2019/9/9d/T--CSU_CHINA--Improved_part.jpg" class="mx-auto" style="width: 80% ">
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</p>
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                        <div class="d-flex justify-content-center">
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</div>
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                                <p style="color: grey; width: 80%; margin-bottom: 32px;text-align: center;">(A) The relative strength of previous part ‘CMV-Gal4-VP16’. (B) The relative strength of the new part ’s(GATA3)p-GAD-miR101-BD’. (C) The comparison of the specificity in cancer cells between new part and the previous part.</p>
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</div>
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                        </div>
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                        <h3>Appendix: Brief introduction of luciferase assay</h3>
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<div class="column full_size">
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                        <p>The Luciferase Assay System is substantially improved over conventional assay methods in both sensitivity and simplicity. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. The Luciferase Assay System incorporates coenzyme A (CoA) for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The Luciferase Assay System yields linear results over at least eight orders of magnitude. Less than 10-20 moles of luciferase have been detected under optimal conditions. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay.</p>
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<h3>Best Part Collection Special Prize</h3>
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                    <div id="References">
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<p> To be eligible for this award, each part in the collection <b>must be well documented on the part's Main Page on the Registry</b>. If you have a collection of parts 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 numbers 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.</p>
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                            <h2>References</h2>
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</div>
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                            <p>[1] H Guan et al.  Int J Mol Med (2016), MicroRNA-101 inhibits cell proliferation and induces apoptosisby targeting EYA1 in breast cancer.</p>
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                            <p>[2] Solberg N1, Krauss S. , Methods Mol Biol. 2013;977:65-78. Luciferase assay to study the activity of a cloned promoter DNA fragment.</p>
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Latest revision as of 00:53, 22 October 2019

loading……

Improved Part

Composite Part
Section Name Type Description
New part BBa_K2908676 DNA s(GATA3)p-GAD-miR101-BS
Existing part BBa_K2580666 DNA CMV-Gal4-VP16

New part

s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]

The part improved in the specificity in Triple negative cancer (TNBC).


Existing part

CMV-Gal4-VP16 (BBa_K2580666)

The part has low specificity in the TNBC cells compared to normal cells although the team members who created the part stated that it is a cancer-specific part.

As for this new part we created this year, it contains the brand new synthetic promoter we designed (BBa_K2908000), the existing sequence in the previous iGEM parts, GAD (Gal4-VP16), and the new designed part miR101-BD.

In our project, we wanted to make the expression of GAD to be specific in Triple negative breast cancer cells (TNBC cells). After communicating with the team creating the part who says that in breast cancer, this part has higher efficiency in cancer cells than normal cells, we tested the efficiency of GAD expression by co-transfecting the CMV-Gal4-VP16 plasmid and pGL4.22-G8p (pGL4.22 has a luciferase gene after G8p, a promoter can be driven by Gal4-VP16) into TNBC cells (cancer cells) and normal cells. A in the figure below shows the results.

Then, we exchange the promoter CMV into s(GATA3)p, and added a miR101-BD in the 3’UTR of the gene GAD (Gal4-VP16), as miR101 is low expressed in the cancer cells while high in the normal cells [1] (the binding of miR101 to miR101-BD can inhibit the expression of GAD protein, then to down-regulate the efficiency of G8p). We used the same method to test the efficiency of our part, that is, co-transfecting the s(GATA3)p-GAD-miR101-BD plasmid and pGL4.22-G8p, and doing the luciferase assay [2]. The figure B shows the results. We also compared the specificity between CMV-Gal4-VP16 and s(GATA3)p-GAD-miR101-BD (C), which indicated that our part dramatically improved the function in specifically regulating the expression of GAD.

(A) The relative strength of previous part ‘CMV-Gal4-VP16’. (B) The relative strength of the new part ’s(GATA3)p-GAD-miR101-BD’. (C) The comparison of the specificity in cancer cells between new part and the previous part.

Appendix: Brief introduction of luciferase assay

The Luciferase Assay System is substantially improved over conventional assay methods in both sensitivity and simplicity. Light is produced by converting the chemical energy of luciferin oxidation through an electron transition, forming the product molecule oxyluciferin. Firefly luciferase, a monomeric 61kDa protein, catalyzes luciferin oxidation using ATP-Mg2+ as a cosubstrate. In the conventional assay for luciferase, a flash of light is generated that decays rapidly after the enzyme and substrates are combined. The Luciferase Assay System incorporates coenzyme A (CoA) for improved kinetics, allowing greater enzymatic turnover and resulting in increased light intensity that is nearly constant for at least 1 minute. The Luciferase Assay System yields linear results over at least eight orders of magnitude. Less than 10-20 moles of luciferase have been detected under optimal conditions. Generally, 100-fold greater sensitivity can be achieved over the chloramphenicol acetyltransferase (CAT) assay.

References

[1] H Guan et al.  Int J Mol Med (2016), MicroRNA-101 inhibits cell proliferation and induces apoptosisby targeting EYA1 in breast cancer.

[2] Solberg N1, Krauss S. , Methods Mol Biol. 2013;977:65-78. Luciferase assay to study the activity of a cloned promoter DNA fragment.

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