Difference between revisions of "Part:BBa K4164998"

Revision as of 14:18, 12 October 2022

ddRFPA1-ddRFPB1

The red fluorescent protein heterodimer is formed by the polymerization of two monomeric proteins ddRFPA1 and ddRFPB1.The monomer is derived from the directed evolution of dTomato gene. ddRFPA1 exhibits weak fluorescence, ddRFPB1 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 ddRFPA1 and ddRFPB1 by a flexible linker(3*GGGGS) to verify the feasicility of ddRFPA1 and ddRFPB1. We cloned this part into the pET-29a+(Figure 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 ddRFPA1 and ddRFPB1 as our report device.

Figure 2. 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.

Figure 7.Comparison of BBa_K4164012 and BBa_K4164016. Left:BBa_K4164012; Right:BBa_K4164016.

Sequence and Features