Difference between revisions of "Part:BBa K4164998"
 
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<partinfo>BBa_K4164998 short</partinfo>
 
<partinfo>BBa_K4164998 short</partinfo>
  
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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.
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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.
  
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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.
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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.
  
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We connected ddRFPA1 and ddRFPB1 by a flexible linker(3*GGGGS) to verify the feasicility of ddRFPA1 and ddRFPB1. 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.
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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 <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 ddRFP-A1 and ddRFP-B1 as our report device.
  
  
 
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<p style="text-align: center;"><img src="https://static.igem.wiki/teams/4164/wiki/part-registry/part-004005016998ddrfpplate.png"with="1000" height="" width="500" height=""/></p>
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<p style="text-align: center;"><img src="https://static.igem.wiki/teams/4164/wiki/project-result/figure2.png"with="1000" height="" width="500" height=""/></p>
 
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<p style="text-align: center!important;"><b>Fig.1 Fluorescence image of <em> E. coli</em> expressing ddRFPA1-ddRFPB1 and control.</b></p>
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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>
  
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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.
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<p style="text-align: center;"><img src="https://static.igem.wiki/teams/4164/wiki/engineering-success/figure-7.png"with="1000" height="" width="500" height=""/></p>
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<p style="text-align: center!important;"><b>Fig. 2. Comparison of BBa_K4164012 and BBa_K4164016.
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Left:BBa_K4164012; Right:BBa_K4164016.</b></p>
  
  

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


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]