Part:BBa_K4046300

DhdO Binding Site #1 with Spacer

This is a binding site for the DhdR gene, as identified in the literature. Through testing, this version of the binding site was characterized as having a K_D value of .64 ± .1 µM, which was one of the lowest values of the different sequences tested. This version of the binding site has an attached spacer sequence, which was included because literature sources indicate that the presence of spacer sequences may impact transcription factor binding.

As part of our goal to construct a biosensor system for the compound D-2-HG, we will utilize a plasmid expressing the DhdR gene (BBa_K4046000) to provide baseline expression of the repressor gene. 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 this 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. 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. For an initial proof-of-concept, we introduced the binding sites into a commercially available pcDNA5 plasmid (Thermo Fischer, V103320) with mCherry fluorescence protein. HEK cells were transfected with these plasmids and imaged for fluorescent activity.

Figure 1: Fluorescent microscopy results of HEK293T cells transfected with our binding site plasmids.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]