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
- 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.
- 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.
- 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.
- 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.
