Difference between revisions of "Part:BBa K4164004"

Latest revision as of 14:11, 13 October 2022

dimerization-dependent red fluorescent protein-A1(ddRFP-A1)

The dimerization-dependent red fluorescent protein-A1 (ddRFP-A1) is a variant of the dimeric Tomato, which is a monomer protein of the red fluorescent protein heterodimer, with a size of 26.4kDa.

ddRFP-A1 is an RFP variant with weak fluorescence derived from the directed evolution of dTomato gene. It has a chromogenic group and can exhibit weak fluorescence in the monomeric state. ddRFP-A1 can form a heterodimer with ddRFP-B1, increasing the fluorescence intensity ten times more than the dissociation state. The utility of ddRFP-A1B1 has been demonstrated in three applications, including the detection of a protein-protein interaction in vitro, imaging of the reversible Ca²⁺-dependent association of calmodulin and imaging of caspase-3 activity during apoptosis.

We connected ddRFP-A1 and ddRFP-B1 by a flexible linker(3*GGGGS) to verify the feasicility of ddRFP-A1 and ddRFP-B1. Following overnight incubation at 37℃, we can see the bright red fluorescence (Fig.1). In this case, we chose ddRFP-A1 and ddRFP-B1 as our report device.

Fig.1 Fluorescence image of E. coli expressing ddRFPA1-ddRFPB1 and control.

In our project, we fused ddRFP-A1 and ddRFP-B1 to the C-terminus of FXR and RXR, respectively. Taking advantage of the characteristics that RXR can bind to the FXR as a heterodimer, using FXR as the receptor, and when the downstream ddRFP-A1 and ddRFP-B1 are correspondingly driven to dimerize and exhibit red fluorescence.

Sequence and Features