Difference between revisions of "Part:BBa K4164005"

Latest revision as of 14:13, 13 October 2022

dimerization-dependent red fluorescent protein-B1(ddRFP-B1)

The dimerization-dependent red fluorescent protein-B1(ddRFP-B1) is a variant of the dimeric Tomato. ddRFP-B1 is a monomer protein of the red fluorescent protein heterodimer, with a size of 26.4kDa, which is a dTomato-derived partner capable of forming dimers with ddRFP-A1. ddRFP-A1 interacts with ddRFP-B1 with a Kd of 33 μM, and, therefore, the heterodimers can exist primarily in a free state at cytoplasmic concentrations sufficient for live cell imaging. ddRFP-B1 does not form a chromatography group and exhibits no 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 use 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, M13 in live cells, 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.

Sequence and Features