Difference between revisions of "Part:BBa K4046870:Design"

Revision as of 20:36, 21 October 2021

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

Design Notes

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

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.

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.