Difference between revisions of "Part:BBa K4164998"

Revision as of 11:51, 13 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(+)(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 ddRFPA1 and ddRFPB1 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1174
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 733
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 7: / Line 7: @@
 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 Ca<sup>2+</sup>-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(+)(Fig.2a) and expressed recombinant proteins in <em>E.coli</em> 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.
+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(+)(Fig. 2a) and expressed recombinant proteins in <em>E.coli</em> 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.
@@ Line 14: / Line 14: @@
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-<p style="text-align: center!important;"><b>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 <em>E.coli</em> expressing ddRFPA1-ddRFPB1 and control.</b></p>
+<p style="text-align: center!important;"><b>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 <em>E.coli</em> expressing ddRFPA1-ddRFPB1 and control.</b></p>
 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.