Difference between revisions of "Part:BBa K4046920:Design"

 
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===Design Notes===
 
===Design Notes===
 
tdTomato is the modified product of a gene originally from mushroom corals in the <i> Discosoma </i> family. The gene was obtained through PCR of a commercially available plasmid pCRISPR-HOT_tdTomato (Addgene, 138567). In normal function, tdTomato is a red fluorescence protein.
 
tdTomato is the modified product of a gene originally from mushroom corals in the <i> Discosoma </i> family. The gene was obtained through PCR of a commercially available plasmid pCRISPR-HOT_tdTomato (Addgene, 138567). In normal function, tdTomato is a red fluorescence protein.
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[[File:T--Duke--DhdrSchematic.png|600 px]] <br>
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<b><font size="+1">Figure 1: This figure outlines the mechanism of the interaction between the DhdR allosteric transcription factor and the <i>dhdO</i> binding site.</font></b>
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This plasmid will be tested together with another construct expressing the protein DhdR, which is a transcriptional repression factor isolated from the bacteria <i>Achromobacter denitrificans</i>. In a wild-type environment, without the presence of DhdR, we expect normal expression of the reporter protein. However, when DhdR is present, it will bind to the <i>dhdO</i> binding site, allosterically blocking the transcription of our reporter gene. When D-2-HG is elevated, particularly in <i>IDH1</i> mutant cells, it binds to DhdR, releasing it from the binding site.
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[[File:T--Duke--T--Duke--DhdO0 curve.png|600 px]] <br>
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<b><font size="+1">Figure 2: This figure outlines the energetics of the interaction between the DhdR allosteric transcription factor and the <i>dhdO</i> binding site.</font></b>
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This allows for transcription of the downstream reporter protein sequence, resulting in brighter expression that is visible in our <i>in vivo</i> droplet system. Since D-2-HG levels are elevated due to the <i>IDH1</i> mutation, we expect that there will be an increase in fluorescence or luminescence due to the release of the allosteric transcription factor caused by the binding of the upregulated oncometabolite. When we perform drug screening assays on our completed co-culture system, we will associate decreased fluorescence or luminescence with lower levels of D-2-HG, which is associated with a variety of downstream metabolic impacts.
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Literature sources indicate that repressor binding can be affected by variables like the number of repeats of the binding site present, as well as the presence of spacer sequences in between repeats of the binding sequence. Because of these variations, we intend to test several different combinations to see which functions the best. By optimizing the binding site and promoter combination, we hope to establish a reporter system that allows for precise quantification of D-2-HG levels over a large dynamic range.
  
  
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===References===
 
===References===
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Xiao, D., Zhang, W., Guo, X., Liu, Y., Hu, C., Guo, S., Kang, Z., Xu, X., Ma, C., Gao, C., & Xu, P. (2021). A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DHDR. https://doi.org/10.1101/2021.02.18.430539
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Cambridge, S. (2014). Photoswitching proteins: Methods and protocols (Vol. 1148). Humana Press.

Revision as of 15:01, 21 October 2021


CMV - BS #1 - BS #1 - Kozak - mCherry - bghA


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1038
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1038
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1038
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1038
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. The gene was obtained through PCR of a commercially available plasmid pCRISPR-HOT_tdTomato (Addgene, 138567). In normal function, tdTomato is a red fluorescence protein.

T--Duke--DhdrSchematic.png
Figure 1: This figure outlines the mechanism of the interaction between the DhdR allosteric transcription factor and the dhdO binding site.

This plasmid will be tested together with another construct expressing the protein DhdR, which is a transcriptional repression factor isolated from the bacteria Achromobacter denitrificans. In a wild-type environment, without the presence of DhdR, we expect normal expression of the reporter protein. However, when DhdR is present, it will bind to the dhdO binding site, allosterically blocking the transcription of our reporter gene. When D-2-HG is elevated, particularly in IDH1 mutant cells, it binds to DhdR, releasing it from the binding site.


T--Duke--T--Duke--DhdO0 curve.png
Figure 2: This figure outlines the energetics of the interaction between the DhdR allosteric transcription factor and the dhdO binding site. 

This allows for transcription of the downstream reporter protein sequence, resulting in brighter expression that is visible in our in vivo droplet system. Since D-2-HG levels are elevated due to the IDH1 mutation, we expect that there will be an increase in fluorescence or luminescence due to the release of the allosteric transcription factor caused by the binding of the upregulated oncometabolite. When we perform drug screening assays on our completed co-culture system, we will associate decreased fluorescence or luminescence with lower levels of D-2-HG, which is associated with a variety of downstream metabolic impacts.

Literature sources indicate that repressor binding can be affected by variables like the number of repeats of the binding site present, as well as the presence of spacer sequences in between repeats of the binding sequence. Because of these variations, we intend to test several different combinations to see which functions the best. By optimizing the binding site and promoter combination, we hope to establish a reporter system that allows for precise quantification of D-2-HG levels over a large dynamic range.


Source

This gene was synthesized commercially from the published sequence (IDT). The DhdR gene is a transcriptional repression factor that is derived from the bacteria Achromobacter denitrificans. The dhdO binding sites were designed and modified to improve binding behavior of the DhdR protein to the sequence.

References

Xiao, D., Zhang, W., Guo, X., Liu, Y., Hu, C., Guo, S., Kang, Z., Xu, X., Ma, C., Gao, C., & Xu, P. (2021). A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DHDR. https://doi.org/10.1101/2021.02.18.430539

Cambridge, S. (2014). Photoswitching proteins: Methods and protocols (Vol. 1148). Humana Press.