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<img src="https://2019.igem.org/wiki/images/thumb/4/44/T--Humboldt_Berlin--YFP_klon.tif/800px-T--Humboldt_Berlin--YFP_klon.tif.jpeg" alt="YFP-clone" width="500" />
 
<img src="https://2019.igem.org/wiki/images/thumb/4/44/T--Humboldt_Berlin--YFP_klon.tif/800px-T--Humboldt_Berlin--YFP_klon.tif.jpeg" alt="YFP-clone" width="500" />
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<caption>Fig. 1 - Fluorescent C. reinhardtii with our construct under a fluorescent microscope.</caption>
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<figcaption>Fig. 1 - Fluorescent C. reinhardtii with our construct under a fluorescent microscope.</figcaption>
  
 
<img class="is-revealing" src="https://2019.igem.org/wiki/images/7/73/T--Humboldt_Berlin--YFP_fluorescence_intensity.png" alt="fluorescence intensity" width="500" />
 
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<caption>Fig. 2 - Fluorescence intensity of C. reinhardtii WT and a YFP carrying clone, in decreasing optical density of the cell culture. Emxcitation at 490 nm and emission measurement at 528 nm.</caption>
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<figcaption>Fig. 2 - Fluorescence intensity of C. reinhardtii WT and a YFP carrying clone, in decreasing optical density of the cell culture. Emxcitation at 490 nm and emission measurement at 528 nm.</figcaption>
  
 
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Revision as of 14:50, 15 October 2019


L1c-Psad-YFP-RbcS2, Expression of YFP through light-inducible promoter

This Level 1 Construct is designed for comparison of locus dependent expression through fluorescence intensity measuerments. The Construct consist of the Psad promoter an coding sequence for the yellow fluorescent protein and the Rbcs2 terminator. The promoter is inducible by light.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1046
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1046
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1046
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1046
    Illegal NgoMIV site found at 1523
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This construct is designed for the expression of the yellow fluorescent protein (YFP) mVenus in C. reinhardtii. It is composed of the PsaD promoter, which stems from the photosystem one complex, the mVenus coding sequence and the terminator Rbcs2, taken from the Rubisco gene. This construct will make C. reinhardtii glow under a fluorescence microscope.

The YFP mVenus has an excitation peak at 515 nm and an emission peak at 528 nm (Kremers et al. 2006). With this construct we focused on the characterization of the light induction of PsaD.

Characterization

The first qualitative characterization we made was to check the fluorescence of C. reinhardtii that had been transformed with this construct under the microscope.

YFP-clone
Fig. 1 - Fluorescent C. reinhardtii with our construct under a fluorescent microscope.
fluorescence intensity
Fig. 2 - Fluorescence intensity of C. reinhardtii WT and a YFP carrying clone, in decreasing optical density of the cell culture. Emxcitation at 490 nm and emission measurement at 528 nm.

References

  1. Kremers, G. J., Goedhart, J., van Munster, E. B., & Gadella, T. W. (2006). Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry, 45(21), 6570-6580.