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

Latest revision as of 21:25, 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

dhdO sequences were designed and optimized for binding to the DhdR protein. Furthermore, a promoter, Kozak sequence, and a terminator were added.

Source

This includes 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.

mCherry is a fluorescent gene product derived from the DsRed gene of mushrooms corals in the Discosoma family. This gene was obtained directly through assembly into the pcDNA5 plasmid (Thermo Fischer, V103320), which contained the mCherry gene. In normal function, mCherry 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.

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.

@@ Line 7: / Line 7: @@
 ===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.
-[[File:T--Duke--DhdrSchematic.png|600 px]] <br>
+<i>dhdO</i> sequences were designed and optimized for binding to the  <i>DhdR</i> protein. Furthermore, a promoter, Kozak sequence, and a terminator were added.
-<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>
-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.
+===Source===
+This includes identified binding site for the <i>DhdR</i> gene, as described by Xiao et al, 2021. 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.
+mCherry is a fluorescent gene product derived from the DsRed gene of mushrooms corals in the <i> Discosoma </i> family. This gene was obtained directly through assembly into the pcDNA5 plasmid (Thermo Fischer, V103320), which contained the <i> mCherry </i> gene. In normal function, mCherry is a red fluorescence protein.
-[[File:T--Duke--T--Duke--DhdO0 curve.png|600 px]] <br>
+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.
-<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 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 <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.
 ===References===