Difference between revisions of "Part:BBa K5195010"
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The 2024 Stanford iGEM team designed a fusion protein consisting of mNeonGreen ([https://parts.igem.org/Part:BBa_K5195004] BBa_K5195004), a P2A linker ([https://parts.igem.org/Part:BBa_K1442039] BBa_K1442039), and a truncated version of the DUX4 protein coding for only the first 217 amino acids (DUX4-DBD) ([https://parts.igem.org/Part:BBa_K5195002] 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. | The 2024 Stanford iGEM team designed a fusion protein consisting of mNeonGreen ([https://parts.igem.org/Part:BBa_K5195004] BBa_K5195004), a P2A linker ([https://parts.igem.org/Part:BBa_K1442039] BBa_K1442039), and a truncated version of the DUX4 protein coding for only the first 217 amino acids (DUX4-DBD) ([https://parts.igem.org/Part:BBa_K5195002] 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. | ||
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This coding sequence was used in a plasmid containing a doxycycline-inducible Tet-On Tight TRE promoter and the coding sequence for the full length DUX4 protein. Primers to amplify the entire plasmid except for the end of the DUX4-fl coding sequence to exclude the transcriptional activation domain. Then, an mNeonGreen-P2A fragment, replicated by PCR from an existing plasmid pK381, provided to us by one of our mentors, Dr. Phillip Kyriakakis, from the Stanford Coleman lab, was gel-purified and then Gibson Assembled with the DUX4-DBD fragment to create our imNG-P2A-DUX4-DBD plasmid. | This coding sequence was used in a plasmid containing a doxycycline-inducible Tet-On Tight TRE promoter and the coding sequence for the full length DUX4 protein. Primers to amplify the entire plasmid except for the end of the DUX4-fl coding sequence to exclude the transcriptional activation domain. Then, an mNeonGreen-P2A fragment, replicated by PCR from an existing plasmid pK381, provided to us by one of our mentors, Dr. Phillip Kyriakakis, from the Stanford Coleman lab, was gel-purified and then Gibson Assembled with the DUX4-DBD fragment to create our imNG-P2A-DUX4-DBD plasmid. | ||
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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. | ||
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The above graph displays the plate reader assay results from 4 replicates of this transfection. It supports the visual results from the fluorescence microscope, showing that with increasing DUX4-DBD to DUX4-fl ratios, the DUX4 reporter expression level is driven down very close to its baseline level. | The above graph displays the plate reader assay results from 4 replicates of this transfection. It supports the visual results from the fluorescence microscope, showing that with increasing DUX4-DBD to DUX4-fl ratios, the DUX4 reporter expression level is driven down very close to its baseline level. | ||
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To obtain more precise, single-cell level data from our transfection experiments, we used flow cytometry to view individual cells and their fluorescence, which gives us a continuum of signals. The flow data supports our idea that as DUX4-DBD:DUX4-fl ratio increases, the corresponding reporter activity in those cells also decreases. | To obtain more precise, single-cell level data from our transfection experiments, we used flow cytometry to view individual cells and their fluorescence, which gives us a continuum of signals. The flow data supports our idea that as DUX4-DBD:DUX4-fl ratio increases, the corresponding reporter activity in those cells also decreases. | ||
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In the above figure, C12, C6, C1, and B8 represent different ratios of DUX4-DBD to DUX4-fl from highest to lowest (C12 is 25:1 DBD:FL, C6 is 10:1, C1 is 1:1, and B8 is 0:1). | In the above figure, C12, C6, C1, and B8 represent different ratios of DUX4-DBD to DUX4-fl from highest to lowest (C12 is 25:1 DBD:FL, C6 is 10:1, C1 is 1:1, and B8 is 0:1). | ||
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These experiments were completed in HEK293T cells, which, though are easy to work with, were not the most representative of the environment our construct is ultimately aimed to be used in. Therefore, we tried to repeat our experiments in C2C12 cells, which is a mouse myoblast cell line. However, we noticed that our transfection efficiency was decreased, and though this was improved after optimization of transfection conditions, it was more difficult to visually see the trend of the DUX4 reporter decreasing with increased DUX4-DBD. The C2C12 transfection visual results are shown below: | These experiments were completed in HEK293T cells, which, though are easy to work with, were not the most representative of the environment our construct is ultimately aimed to be used in. Therefore, we tried to repeat our experiments in C2C12 cells, which is a mouse myoblast cell line. However, we noticed that our transfection efficiency was decreased, and though this was improved after optimization of transfection conditions, it was more difficult to visually see the trend of the DUX4 reporter decreasing with increased DUX4-DBD. The C2C12 transfection visual results are shown below: | ||
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Our fluorescence plate reader assay for the C2C12 transfection demonstrated the same trend of increasing DUX4-DBD decreasing DUX4-fl binding: | Our fluorescence plate reader assay for the C2C12 transfection demonstrated the same trend of increasing DUX4-DBD decreasing DUX4-fl binding: | ||
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Revision as of 19:40, 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:
