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-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 |
