Difference between revisions of "Part:BBa K4046990"

Latest revision as of 02:28, 22 October 2021

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

Overview This is a binding site for the DhdR gene, as identified in the literature by Xiao et al. The DhdR protein was originally identified in Achromobactor denitrificans, and the dhdO sequences were designed and optimized for binding to the DhdR protein. In this composite part, we added a Kozak sequence and mCherry gene downstream of the binding site and promoter, which allow the part to be used in a DhdR-based biosensor for oncometabolite D-2-HG.

Design This composite part is one combination of our D-2-HG reporter collection. A schematic for the operation of our system is shown below.

Figure 1: 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.

The derepression of the circuit 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 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.

In addition, results from our mathematical model show that the activation relationship between D-2-HG and reporter expression becomes less steep with increasing independent binding sites.

Data

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 3: Fluorescent microscopy verification of the expression of the dhdO binding sites inserted into a pcDNA5 backbone tagged with mCherry, expressed in HEK293T cells. Expression of the pcDNA5 plasmid with no insert is included as a positive control and expression of unverified BS3 plasmid as a negative control.

Sequence and Features