Part:BBa_K4046810:Design
CMV - BS #1 - BS #1 - Kozak - cLuc - bghA
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 614
Illegal BglII site found at 1278
Illegal BglII site found at 1337 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
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. 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. cLuc is an enzyme isolated from the genomic sequence of V. hilgendorfii , a marine crustacean. This gene was obtained through PCR of a commercially available plasmid pIND-CLucZ (Addgene, 53224). In normal function, cLuc is a luciferase enzyme that produces luminescence upon contact with its substrate, vargulin.
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.