Difference between revisions of "Team:CSU CHINA/Part Collection"
Lonely-boy (Talk | contribs) |
Lonely-boy (Talk | contribs) |
||
| (9 intermediate revisions by the same user not shown) | |||
| Line 30: | Line 30: | ||
cursor: pointer; | cursor: pointer; | ||
} | } | ||
| − | img { | + | .container img{ |
display: block; | display: block; | ||
margin: 0 auto; | margin: 0 auto; | ||
| + | box-shadow: 5px 5px 10px #888888; | ||
} | } | ||
.Teenav{ | .Teenav{ | ||
| Line 46: | Line 47: | ||
.Teenav li{ | .Teenav li{ | ||
margin:10px 0px; | margin:10px 0px; | ||
| + | font-size:1rem!important; | ||
} | } | ||
.Teenav a{ | .Teenav a{ | ||
| Line 57: | Line 59: | ||
#toTOP{ | #toTOP{ | ||
position:fixed; | position:fixed; | ||
| − | right: | + | right:2%; |
| − | bottom: | + | bottom:2%; |
border-radius:25%; | border-radius:25%; | ||
} | } | ||
| Line 71: | Line 73: | ||
</style> | </style> | ||
<div class="container-fluid Topbox" id="Top"> | <div class="container-fluid Topbox" id="Top"> | ||
| − | <h1>Improved Part</h1> | + | <h1 class="display-3">Improved Part</h1> |
</div> | </div> | ||
<div class="container"> | <div class="container"> | ||
<div class="row"> | <div class="row"> | ||
<div id="col-md-8 offset-md-2"> | <div id="col-md-8 offset-md-2"> | ||
| − | <div id="mRNA" style="width: 100%; margin-bottom: 30px;"> | + | <div id="mRNA" style="width: 100%; margin-top:30px;margin-bottom: 30px;"> |
<div class="card"> | <div class="card"> | ||
<div class="card-header"> | <div class="card-header"> | ||
| − | <a class="card-link" data-toggle="collapse" href="#mRNA1" style="color: black"> | + | <a class="card-link" data-toggle="collapse" href="#mRNA1" style="color: black">Composite Part</a> |
</div> | </div> | ||
| − | <div id="mRNA1" class="collapse" data-parent="#mRNA"> | + | <div id="mRNA1" class="collapse show" data-parent="#mRNA"> |
<div class="card-body"> | <div class="card-body"> | ||
<figure> | <figure> | ||
| Line 105: | Line 107: | ||
<td>Existing part</td> | <td>Existing part</td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K2580666">BBa_K2580666</td> | <td><a href="http://parts.igem.org/Part:BBa_K2580666">BBa_K2580666</td> | ||
| − | <td> | + | <td>DNA</td> |
<td>CMV-Gal4-VP16</td> | <td>CMV-Gal4-VP16</td> | ||
</tr> | </tr> | ||
| Line 119: | Line 121: | ||
| − | + | ||
| − | + | ||
<h3>New part</h3> | <h3>New part</h3> | ||
| − | <img src="./ | + | <img src="https://static.igem.org/mediawiki/2019/7/77/T--CSU_CHINA--Improved_part.png" class="mx-auto" style="width: 80% "> |
<p class="ts"><b>s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]</b></p> | <p class="ts"><b>s(GATA3)p-GAD-miR101-BD (BBa_K2908676) [GAD is totally the same as Gal4-VP16]</b></p> | ||
<div class="d-flex justify-content-center"> | <div class="d-flex justify-content-center"> | ||
| Line 129: | Line 131: | ||
<br> | <br> | ||
<h3>Existing part</h3> | <h3>Existing part</h3> | ||
| − | <img src="./ | + | <img src="https://static.igem.org/mediawiki/2019/0/00/T--CSU_CHINA--CMV-Gal4-VP16.png" class="mx-auto" style="width: 80% "> |
<p class="ts"><b>CMV-Gal4-VP16 (BBa_K2580666)</b></p> | <p class="ts"><b>CMV-Gal4-VP16 (BBa_K2580666)</b></p> | ||
<div class="d-flex justify-content-center"> | <div class="d-flex justify-content-center"> | ||
| Line 138: | Line 140: | ||
<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> | <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> | ||
<p></p> | <p></p> | ||
| − | <img src="./ | + | <img src="https://static.igem.org/mediawiki/2019/9/9d/T--CSU_CHINA--Improved_part.jpg" class="mx-auto" style="width: 80% "> |
<div class="d-flex justify-content-center"> | <div class="d-flex justify-content-center"> | ||
<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> | <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> | ||
Latest revision as of 00:53, 22 October 2019
loading……
Improved 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.