Difference between revisions of "Part:BBa K4164998"

Latest revision as of 14:16, 13 October 2022

ddRFPA1-ddRFPB1

The red fluorescent protein heterodimer is formed by the polymerization of two monomeric proteins ddRFP-A1 and ddRFP-B1.The monomer is derived from the directed evolution of dTomato gene. ddRFP-A1 exhibits weak fluorescence, ddRFP-B1 has no fluorescence. 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. We cloned this part into the pET-29a(+)(Fig. 2a) and expressed recombinant proteins in E.coli BL21(DE3). 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. a. Results for pET29a(+)-ddRFPA1-B1. Lane2, lane 4, the whole length of these plasmids is 6594bp. b. The plasmid map of pET29a(+)-ddRFPA1-B1. c, d. Fluorescence image of E.coli expressing ddRFPA1-ddRFPB1 and control.

Moreover, we constructed 2 ddRFPA1-ddRFPB1 expression plasmids with different promoters (BBa_K4164012, BBa_K4164016) to optimize expression conditions. The result showed that T7-ddRFPA1-ddRFPB1 (BBa_K4164016) was capable of emitting visible fluorescence with very high intensity.

Fig. 2. Comparison of BBa_K4164012 and BBa_K4164016. Left:BBa_K4164012; Right:BBa_K4164016.

Sequence and Features