Basic part
Abstract
For this year’s iGEM competition, our preferable basic parts are AR185-T2A -EGFP (BBa_K2865001) and BNP promotor (BBa_K2865000) .
Basic part--AR185
The part of AR185 is a coding which can be translated for one of the RyR2-specific nanobodies isolated from a phage display library of variable domains of camellidae heavy chain-only antibodies (VHH).With the feature of inhibiting PKA dependent S2808 phosphorylation on RyR2 in an vitro assay, it was chosen for further investigation on the treatment of heart failure. To evaluate its potential use for the treatment of heart failure, an adeno-associated virus (AAV) based intracellular antibody delivery strategy were adopt to achieve cardiac-specific gene-therapy and demonstrated therapeutic effect both in cell-based assays and in vivo models. The construct of the intracellular antibody fragment, AR185, was engineered to express an upstream EGFP reporter separated from the nanobody sequence by a T2A sequence. The sequence encoding the whole fragments construct was subcloned into the pAAV vector resulting in AAV.AR185.
Figure 1. Isolation of RyR2-specific nanobody by phage display.
(A) Phage-displayed nanobody fragments were selected against RyR2 by four rounds of panning. A gradual increase in phage titers was detected after each round of panning. (B) Polyclonal phage ELISA from the output phage of each round of panning. Control group used BSA as the irrelevant antigen. (C) Heat map generated from ELISA data of purified RyR2 channels which were phosphorylated in the presence of the PKA. (D) Kinetic analysis of AR185 binding to RyR2 was performed by SPR.
Basic part--BNP promotor
The part of BNP promotor is a regulator which can be used to control gene expression in heart failure gene therapy. Because BNP, known as B-type natriuretic peptide, has been regarded as an important biomarker in the diagnosis of heart failure (HF), whose level remains low in normal hearts and elevates dramatically in the blood of patients with HF. What’s more, BNP promoter activity is related to the severity of HF. Therefore, our team wants to utilize the ability that BNP promoter can be activited in the HF environment in order to regulate the related sequences expression for heart failure. Our team propose a promising therapeutic approach for heart failure, Ca2+RTIN.
See More
Integrated information about BNP promotor and AR185 posted on Demonstrate page. Or you can alternatively check the (BBa_K2865000 and BBa_K2865001) on the Registry of Standard Biological Parts website. We listed all the basic parts we constructed this year below.
We listed Biobricks belong to Part Collection below.
| Favourite | Name | Type | Description | Length |
| BBa_K2865001 | Coding | AR185-T2A-EGFP, nanobody inhibiting RyR2 phosphorylation | 1204 | |
| BBa_K2865000 | Regulatory | BNP promoter, heart failure inducible | 508 | |
| BBa_K2865002 | Project | [AAV9]-Left-ITR | 175 | |
| BBa_K2865003 | Project | [AAV9]-Right-ITR | 207 | |
| BBa_K2865004 | Terminator | SV40 poly(A) | 261 |
