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

 
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<h2 class="inner-h">Favorite composite part——BNP-AR185 T2A eGFP-polyA</h2>
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<h3 class="inner-h">Favorite composite part——Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR</h3>
 
<p class="inner-text">
 
<p class="inner-text">
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For this year’s iGEM competition, our favorite composite part is B185, short for BNP-AR185-polyA, This part is composed of BNP promotor, AR185 linked with eGFP by T2A and poly A.
+
For this year's iGEM competition, our favorite composite part is Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR.
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</p>
 
</p>
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<p class="inner-text">
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This device is a shuttle plasmid of the AAV helper-free system. It consists of a CMV promoter, nanobody AR185-T2A-EGFP (BBa_K2865001), SV40 polyA signal and flanked by two inverted terminal repeats (BBa_K2865002 and BBa_K2865003).
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The use of this part is 1) to produce AAV9 particles, 2) to express our novel intrabody AR185 linked with reporter EGFP in failing heart, and 3) to test its efficacy for heart failure.
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</p>
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<div style="text-align:center" class="resultimage">
 
<div style="text-align:center" class="resultimage">
 
<img src="https://static.igem.org/mediawiki/2018/e/e6/T--SMMU-China--Composite_part_Pic_1.png" style="width: 60%;">
 
<img src="https://static.igem.org/mediawiki/2018/e/e6/T--SMMU-China--Composite_part_Pic_1.png" style="width: 60%;">
 
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<p style="font-style: italic;text-align: center;padding: 0em 100px 1em;">
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<strong>Figure 1.The B185 construct</strong>
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<strong>Figure 1. Schematic diagram of constructs of Left ITR-BNP-AR185-T2A-EGFP- Poly(A)-Right ITR</strong>
 
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<h2 class="inner-h">How B185 works</h2>
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<h3 class="inner-h">How it works</h3>
 
<p class="inner-text">
 
<p class="inner-text">
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As has shown presiously, BNP promoter activity is related to the severity of heart failure. So we want to use ET-1, one stimulus factor, to imitate the environment of heart failure and induce BNP promoter expression. Then, the later sequence of the part, AR185, also can be expressed and fuction what it could specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro.  
+
As has shown previously, BNP promoter activity is related to the severity of heart failure. So we want to use ET-1 and AngⅡ, two stimulus factor, to imitate the environment of heart failure and induce BNP promoter expression. To our dismay, BNP promoter didn't response well to HF-related factors AngⅡand ET-1 in our vitro cell experiments, and its function remains to be investigated further. However, the composite part, C185, which is used to package AAV9 containing our therapeutic gene AR185 driven by CMV promoter, has shown the successful results. We have tested and results suggested that AR185 was effective and there were significant differences between AR185 group and control group. Then, the later sequence of the part, AR185, also can be expressed and fuction what it could specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro.
 
</p>
 
</p>
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<div style="text-align:center" class="resultimage">
 
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<img src="https://static.igem.org/mediawiki/2018/a/a1/T--SMMU-China--Part_Collection_Pic_1.jpg" style="width: 60%;">
 
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<p style="font-style: italic;text-align: center;padding: 0em 100px 1em;">
 
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<strong>Figure 2.Design and Function of Ca<sup>2+</sup>RTIN</strong>
 
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</p>
 
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</div>
 
 
 
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<h2 class="inner-h">See More</h2>
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<h3 class="inner-h">See More</h3>
 
<p class="inner-text">
 
<p class="inner-text">
−
Integrated information about AR185 and BNP promotor posted on Demonstrate page. Or you can alternatively check the (BBa_I712008) on the Registry of Standard Biological Parts website.  
+
Integrated information about AR185-T2A-EGFP and BNP promotor posted on Demonstrate page. Or you can alternatively check the the (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865013" target="_Blank">BBa_K2865013</a>) on the Registry of Standard Biological Parts website.  
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<td><i class="fa fa-heart" style="color: #F40"></i></td>
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<td></td>
 
<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865009" target="_Blank">BBa_K2865009</a></td>
 
<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865009" target="_Blank">BBa_K2865009</a></td>
 
<td>Composite</td>
 
<td>Composite</td>
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<td>BNP-AR185-Poly(A)</td>
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<td>BNP-AR185-T2A-EGFP-Poly(A)</td>
 
<td>1989</td>
 
<td>1989</td>
 
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865010" target="_Blank">BBa_K2865010</a></td>
 
<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865010" target="_Blank">BBa_K2865010</a></td>
 
<td>Composite</td>
 
<td>Composite</td>
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<td>CMV-AR185-Poly(A)</td>
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<td>CMV-AR185-T2A-EGFP-Poly(A)</td>
 
<td>2135</td>
 
<td>2135</td>
 
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865012" target="_Blank">BBa_K2865012</a></td>
 
<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865012" target="_Blank">BBa_K2865012</a></td>
 
<td>Composite</td>
 
<td>Composite</td>
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<td>Left ITR-BNP-AR185-Poly(A)-Right ITR</td>
+
<td>Left ITR-BNP-AR185-T2A-EGFP-Poly(A)-Right ITR</td>
 
<td>2387</td>
 
<td>2387</td>
 
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<tr>
 
<tr>
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<td></td>
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<td><i class="fa fa-heart" style="color: #F40"></i></td>
 
<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865013" target="_Blank">BBa_K2865013</a></td>
 
<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2865013" target="_Blank">BBa_K2865013</a></td>
 
<td>Composite</td>
 
<td>Composite</td>
−
<td>Left ITR-CMV-AR185-Poly(A)-Right ITR</td>
+
<td>Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR</td>
 
<td>2533</td>
 
<td>2533</td>
 
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</center>
 
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<h3 class="inner-h">References</h3>
 
−
<ol>
 
−
<li>Sergeeva, I.A. and V.M. Christoffels, Regulation of expression of atrial and brain natriuretic peptide, biomarkers for  heart development and disease. Biochim Biophys Acta, 2013. 1832(12): p. 2403-13.</li>
 
−
<li>Bingham, A.J., et al., The repressor element 1-silencing transcription factor regulates heart-specific gene expression using multiple chromatin-modifying complexes. Mol Cell Biol, 2007. 27(11): p. 4082-92.</li>
 
−
<li>Kerkela, R., et al., Key roles of endothelin-1 and p38 MAPK in the regulation of atrial stretch response. Am J Physiol Regul Integr Comp Physiol, 2011. 300(1): p. R140-9.</li>
 
−
<li>Lu, Y.M., et al., DY-9760e inhibits endothelin-1-induced cardiomyocyte hypertrophy through inhibition of CaMKII and ERK activities. Cardiovasc Ther, 2009. 27(1): p. 17-27.</li>
 
−
</ol>
 
 
</article>
 
</article>
  
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Latest revision as of 16:02, 20 November 2018

Composite part

Favorite composite part——Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR

For this year's iGEM competition, our favorite composite part is Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR.

This device is a shuttle plasmid of the AAV helper-free system. It consists of a CMV promoter, nanobody AR185-T2A-EGFP (BBa_K2865001), SV40 polyA signal and flanked by two inverted terminal repeats (BBa_K2865002 and BBa_K2865003).

The use of this part is 1) to produce AAV9 particles, 2) to express our novel intrabody AR185 linked with reporter EGFP in failing heart, and 3) to test its efficacy for heart failure.

Figure 1. Schematic diagram of constructs of Left ITR-BNP-AR185-T2A-EGFP- Poly(A)-Right ITR

How it works

As has shown previously, BNP promoter activity is related to the severity of heart failure. So we want to use ET-1 and AngⅡ, two stimulus factor, to imitate the environment of heart failure and induce BNP promoter expression. To our dismay, BNP promoter didn't response well to HF-related factors AngⅡand ET-1 in our vitro cell experiments, and its function remains to be investigated further. However, the composite part, C185, which is used to package AAV9 containing our therapeutic gene AR185 driven by CMV promoter, has shown the successful results. We have tested and results suggested that AR185 was effective and there were significant differences between AR185 group and control group. Then, the later sequence of the part, AR185, also can be expressed and fuction what it could specifically bind to RyR2 in rat cardiomyocytes and have the ability to inhibit PKA dependent S2808 phosphorylation in vitro.

See More

Integrated information about AR185-T2A-EGFP and BNP promotor posted on Demonstrate page. Or you can alternatively check the the (BBa_K2865013) on the Registry of Standard Biological Parts website.

We listed other composite part we constructed this year below.

Favourite Name Type Description Length
BBa_K2865006 Composite CMV promoter-eGFP-poly(A) 1666
BBa_K2865008 Composite BNP promoter-eGFP-poly(A) 1520
BBa_K2865009 Composite BNP-AR185-T2A-EGFP-Poly(A) 1989
BBa_K2865010 Composite CMV-AR185-T2A-EGFP-Poly(A) 2135
BBa_K2865012 Composite Left ITR-BNP-AR185-T2A-EGFP-Poly(A)-Right ITR 2387
BBa_K2865013 Composite Left ITR-CMV-AR185-T2A-EGFP-Poly(A)-Right ITR 2533
BBa_K2865014 Composite Left ITR-CMV-eGFP-Poly(A)-Right ITR 2064
BBa_K2865015 Composite Left ITR-BNP-eGFP-Poly(A)-Right ITR 1918
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