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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<h2 class="inner-h">Favorite composite part——CMV-AR185-T2A -eGFP-polyA</h2>
 
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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.
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For this year’s iGEM competition, our favorite composite part is C185, short for CMV-AR185-T2A -eGFP-polyA, This part is composed of CMV promotor, AR185 linked with eGFP by T2A and poly A.
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<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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<strong>Figure 1.The B185 construct</strong>
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<strong>Figure 1. The C185 construct</strong>
 
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<h2 class="inner-h">How  B185 works</h2>
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<h2 class="inner-h">How  C185 works</h2>
 
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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.  
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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.
 
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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%;">
 
<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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<strong>Figure 2.Design and Function of Ca<sup>2+</sup>RTIN</strong>
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<strong>Figure 2. Design and Function of part of Left ITR-CMV-AR185-Poly(A)-Right ITR</strong>
 
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<h2 class="inner-h">See More</h2>
 
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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.  
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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><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>
 
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<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>

Revision as of 07:36, 14 October 2018

Composite part

Favorite composite part——CMV-AR185-T2A -eGFP-polyA

For this year’s iGEM competition, our favorite composite part is C185, short for CMV-AR185-T2A -eGFP-polyA, This part is composed of CMV promotor, AR185 linked with eGFP by T2A and poly A.

Figure 1. The C185 construct

How C185 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.

Figure 2. Design and Function of part of Left ITR-CMV-AR185-Poly(A)-Right ITR

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-Poly(A) 1989
BBa_K2865010 Composite CMV-AR185-Poly(A) 2135
BBa_K2865012 Composite Left ITR-BNP-AR185-Poly(A)-Right ITR 2387
BBa_K2865013 Composite Left ITR-CMV-AR185-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

References

  1. 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.
  2. 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.
  3. 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.
  4. 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.
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