Difference between revisions of "Part:BBa K4046770:Design"
 
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===Design Notes===
 
===Design Notes===
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This gene was synthesized commercially from the published sequence (IDT). tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. 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). 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.
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This composite part is one of several that the Duke iGEM intends to test for use in a biosensor for the oncometabolite D-2-HG. A schematic for the operation of our system is shown below.
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+
  
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[[File:T--Duke--DhdrSchematic.png|600 px]] <br>
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<b><font size="+1">Figure 2: 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.
  
 +
[[File:T--Duke--T--Duke--DhdO5 curve.png|600 px]] <br>
 +
<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>
  
 +
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.
  
 +
Literature sources indicate that repressor binding can be affecting 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===
 
===Source===
  
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This is an identified binding site for the DhdR gene, as described by Xiao et al, 2021. 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. 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. CMV is a constitutive reporter associated with the cytomegalovirus. This gene was obtained through the pcDNA5 backbone that we were using (Thermo Fischer, V103320). In normal function, the CMV promoter allows for high levels of expression of associated gene products.  
+
This gene was synthesized commercially from the published sequence (IDT). The <i>DhdR</i> gene is a transcriptional repression factor that is derived from the bacteria <i>Achromobacter denitrificans</i>. The <i>dhdO</i> binding sites were designed and modified to improve binding behavior of the DhdR protein to the sequence. tdTomato is the modified product of a gene originally from mushroom corals in the <i> Discosoma </i> family. CMV is a constitutive reporter associated with the cytomegalovirus. This gene was obtained through the pcDNA5 backbone that we were using (Thermo Fischer, V103320). In normal function, the CMV promoter allows for high levels of expression of associated gene products. 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.
−
 
+
  
 
===References===
 
===References===
−
A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR
+
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  
−
Dan Xiao, Wen Zhang, Xiaoting Guo, Yidong Liu, Chunxia Hu, Shiting Guo, Zhaoqi Kang, Xianzhi Xu, Cuiqing Ma, Chao Gao, Ping Xu
+
−
bioRxiv 2021.02.18.430539; doi: https://doi.org/10.1101/2021.02.18.430539
+
  
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Sidney Cambridge (ed.), Photoswitching Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1148, DOI 10.1007/978-1-4939-0470-9_7, © Springer Science+Business Media New York 2014
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Cambridge, S. (2014). Photoswitching proteins: Methods and protocols (Vol. 1148). Humana Press.

Latest revision as of 15:11, 21 October 2021


CMV - BS #2 with spacer - BS #2 - Kozak - tdTomato - bghA


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1025
    Illegal PstI site found at 1751
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This composite part is one of several that the Duke iGEM intends to test for use in a biosensor for the oncometabolite D-2-HG. A schematic for the operation of our system is shown below.

T--Duke--DhdrSchematic.png
Figure 2: 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--DhdO5 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 affecting 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. tdTomato is the modified product of a gene originally from mushroom corals in the Discosoma family. CMV is a constitutive reporter associated with the cytomegalovirus. This gene was obtained through the pcDNA5 backbone that we were using (Thermo Fischer, V103320). In normal function, the CMV promoter allows for high levels of expression of associated gene products. 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.

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.