Difference between revisions of "Part:BBa K5195010"
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For testing this device, we first conducted a series of transfections HEK293T cells, seeded at a density of 100,000 cells/well in 24-well plates. Varying ratios of DUX4-DBD to DUX4 full length (DUX4-fl) were transfected alongside a DUX4 reporter that produces mScarlet (red)fluorescent protein upon the activation of the DUX4 binding sites located upstream of the fluorescent protein in the plasmid. 48 hours, the plates were imaged using a fluorescent microscope and a fluorescence plate reader assay was performed. This transfection tested the potential for DUX4-DBD to act as a competitive inhibitor, because with more DUX4-DBD added, we expect the mScarlet fluorescent signal produced by the reporter to decrease because DUX4-DBD binds to the same region of DNA as DUX4-fl, but lacks the transcriptional activation domain responsible for recruiting transcriptional machinery to activate downstream gene expression. Our transfection results are shown below: | For testing this device, we first conducted a series of transfections HEK293T cells, seeded at a density of 100,000 cells/well in 24-well plates. Varying ratios of DUX4-DBD to DUX4 full length (DUX4-fl) were transfected alongside a DUX4 reporter that produces mScarlet (red)fluorescent protein upon the activation of the DUX4 binding sites located upstream of the fluorescent protein in the plasmid. 48 hours, the plates were imaged using a fluorescent microscope and a fluorescence plate reader assay was performed. This transfection tested the potential for DUX4-DBD to act as a competitive inhibitor, because with more DUX4-DBD added, we expect the mScarlet fluorescent signal produced by the reporter to decrease because DUX4-DBD binds to the same region of DNA as DUX4-fl, but lacks the transcriptional activation domain responsible for recruiting transcriptional machinery to activate downstream gene expression. Our transfection results are shown below: | ||
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Here, the red fluorescent protein, our DUX4 reporter readout, decreases with increasing ratios of DUX4-DBD to DUX4-fl. The increasing amount of DUX4-DBD is implied from the increasing mNeonGreen (GFP) signal. | Here, the red fluorescent protein, our DUX4 reporter readout, decreases with increasing ratios of DUX4-DBD to DUX4-fl. The increasing amount of DUX4-DBD is implied from the increasing mNeonGreen (GFP) signal. | ||
Revision as of 19:38, 1 October 2024
mNG-P2A-DUX4-DBD
The 2024 Stanford iGEM team designed a fusion protein consisting of mNeonGreen ([1] BBa_K5195004), a P2A linker ([2] BBa_K1442039), and a truncated version of the DUX4 protein coding for only the first 217 amino acids (DUX4-DBD) ([3] BBa_K5195002). This design was created in order be able to quantify how much DUX4-DBD protein would be produced in cells upon transfection. Because the P2A linker causes ribosomal “skipping” during translation, this effectively separates the mNeonGreen and DUX4-DBD proteins. In designing the coding sequence in this way, we enabled quantification of DUX4-DBD without directly attaching another large protein to it.
Experimental Approach
For testing this device, we first conducted a series of transfections HEK293T cells, seeded at a density of 100,000 cells/well in 24-well plates. Varying ratios of DUX4-DBD to DUX4 full length (DUX4-fl) were transfected alongside a DUX4 reporter that produces mScarlet (red)fluorescent protein upon the activation of the DUX4 binding sites located upstream of the fluorescent protein in the plasmid. 48 hours, the plates were imaged using a fluorescent microscope and a fluorescence plate reader assay was performed. This transfection tested the potential for DUX4-DBD to act as a competitive inhibitor, because with more DUX4-DBD added, we expect the mScarlet fluorescent signal produced by the reporter to decrease because DUX4-DBD binds to the same region of DNA as DUX4-fl, but lacks the transcriptional activation domain responsible for recruiting transcriptional machinery to activate downstream gene expression. Our transfection results are shown below:
Here, the red fluorescent protein, our DUX4 reporter readout, decreases with increasing ratios of DUX4-DBD to DUX4-fl. The increasing amount of DUX4-DBD is implied from the increasing mNeonGreen (GFP) signal.
