Difference between revisions of "Part:BBa K1688004"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1688004 short</partinfo>
 
<partinfo>BBa_K1688004 short</partinfo>
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Composite part consisting of the RBS, BBa_B0034, and the red fluorescent protein, dTomato (BBa_K1688019). dTomato has not been bricked previously.
  
 
<partinfo>BBa_K1688004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1688004 SequenceAndFeatures</partinfo>
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[[File:uppsala_dtomato.jpg]]
 
[[File:uppsala_dtomato.jpg]]
  
−
Figure 1. dTomato expressed in E. coli DH5-alpha with an  
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Figure 1. dTomato expressed in ''E. coli'' DH5-α with an  
 
inducible promoter.
 
inducible promoter.
 +
 +
[[File:uppsala_nahrinduction-dTomato.png]]
 +
 +
Figure 2. dTomato expressed in ''E. coli'' DH5-alpha. NahR/Psal induction test in LB medium. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.
 +
 +
[[File:uppsala_nahrinductionplate123s-dTomato.png]]
 +
 +
Figure 3. dTomato expressed in ''E. coli'' DH5-alpha. NahR/Psal induction test on agar plate. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.
  
  
 
== Usage and Biology==
 
== Usage and Biology==
  
−
dTomato is derived from a monomeric mutant of the anthozoan coral RFP, DsRed. The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use – especially in self-made fluorometry tests – because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm (Figure 2).
+
The dTomato protein is a fluorescent protein dimer, created by direct evolution of the wild-type DsRed, from Discosoma sp. (Shaner et al, - Improved monomeric red, orange and yellow fluorescent proteins derived from ''Discosoma sp.'' red fluorescent protein, 2004). The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use – especially in self-made fluorometry tests – because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm (Figure 4).
  
 
                           [[File:Uppsala2015_dTomato_Chromatogram.png]]
 
                           [[File:Uppsala2015_dTomato_Chromatogram.png]]
−
Figure 2. Excitation (blue curve) and emission (red curve) spectra for the dTomato fluorescent protein (the graph is designed with the following tool: https://www.chroma.com/spectra-viewer)
+
Figure 4. Excitation (blue curve) and emission (red curve) spectra for the dTomato fluorescent protein (the graph is designed with the following tool: https://www.chroma.com/spectra-viewer)
 +
 
 +
== Characterization ==
 +
 
 +
We characterized dTomato by using the NahR/Psal promoter system on a pSB1C3 plasmid (See figure 1,2 and 3). The cells containing the plasmid were streaked on agar plates with different salicylate concentrations in the agar medium. Similarly, we also examined liquid cultures with different concentrations of salicylate in the solution. Both of these tests showed that the bacteria were producing the dTomato protein in presence of salicylate, independent of the salicylate being in liquid culture or in the solid growth medium. It can also be easily observed that the fluorescence increased with increasing salicylate concentrations (See figure 1 and 2). The measurements were made with  the fluorometer that our team built in order to be able to characterize this system. These results were compared to MACS measurements on the same colonies and the measurements were correlated.
 +
 
  
−
dTomato is derived from a monomeric mutant of the anthozoan coral RFP, DsRed.
+
== References ==
 +
Nathan C Shaner, Robert E Campbell, Paul A Steinbach, Ben N G Giepmans, Amy E Palmer & Roger Y Tsien. “Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein”, 2004

Latest revision as of 02:09, 19 September 2015

dTomato (inc RBS)

Composite part consisting of the RBS, BBa_B0034, and the red fluorescent protein, dTomato (BBa_K1688019). dTomato has not been bricked previously.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Uppsala dtomato.jpg

Figure 1. dTomato expressed in E. coli DH5-α with an inducible promoter.

Uppsala nahrinduction-dTomato.png

Figure 2. dTomato expressed in E. coli DH5-alpha. NahR/Psal induction test in LB medium. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.

Uppsala nahrinductionplate123s-dTomato.png

Figure 3. dTomato expressed in E. coli DH5-alpha. NahR/Psal induction test on agar plate. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.


Usage and Biology

The dTomato protein is a fluorescent protein dimer, created by direct evolution of the wild-type DsRed, from Discosoma sp. (Shaner et al, - Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, 2004). The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use – especially in self-made fluorometry tests – because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm (Figure 4). 

                         Uppsala2015 dTomato Chromatogram.png

Figure 4. Excitation (blue curve) and emission (red curve) spectra for the dTomato fluorescent protein (the graph is designed with the following tool: https://www.chroma.com/spectra-viewer)

Characterization

We characterized dTomato by using the NahR/Psal promoter system on a pSB1C3 plasmid (See figure 1,2 and 3). The cells containing the plasmid were streaked on agar plates with different salicylate concentrations in the agar medium. Similarly, we also examined liquid cultures with different concentrations of salicylate in the solution. Both of these tests showed that the bacteria were producing the dTomato protein in presence of salicylate, independent of the salicylate being in liquid culture or in the solid growth medium. It can also be easily observed that the fluorescence increased with increasing salicylate concentrations (See figure 1 and 2). The measurements were made with the fluorometer that our team built in order to be able to characterize this system. These results were compared to MACS measurements on the same colonies and the measurements were correlated.


References

Nathan C Shaner, Robert E Campbell, Paul A Steinbach, Ben N G Giepmans, Amy E Palmer & Roger Y Tsien. “Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein”, 2004