Part:BBa_K4046100
DhdO Binding Site #1
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 KD value of .64 ± .1 µM, which was one of the lowest values of the different sequences tested.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Toulouse-INSA-UPS’s 2023 contribution for the characterization of this dhdO part
As characterized above in part BBa_K4046100 described by iGEM21_Duke (dated October 18, 2021), we used this DhdO Binding Site for our project and contributed to its characterization. Toulouse-INSA-UPS 2023 created part BBa_K4768001 that functions as an inducible promoter and characterized it in PURE system. The team showed that DhdR binds to the operator site dhdO, preventing transcription of the downstream gene. Repression was relieved upon binding of 2-hydroxyglutarate to DhdR. Regulation of gene expression was assessed using the reporter protein sfGFP by fluorescence measurements.
Characterisation
Cell-free production of sfGFP
We used the PCR products of tymp, sfgfp, and anti-HER2 nanobody (anti-HER2-nb) as templates for expression with the PUREfrex2.0 kit (See the protocol <a href="https://2023.igem.wiki/toulouse-insa-ups/protocols">here</a>). Additionally, we supplemented the reaction with GreenLys reagent for the co-translational incorporation of fluorescent lysine residues, which facilitated the detection of synthesized proteins by SDS-PAGE.
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<figcaption class="normal">Figure 3: SDS-PAGE analysis of the gene expression products (Mini-PROTEAN TGX Stain-free Gels). The overlay of the GreenLys and stain-free images are shown. Lanes from left to right: negative control without DNA, positive control with <i>dhfr control plasmid, TYMP with an expected size of 52 kDa, sfGFP with an expected size of 26 kDa, anti-HER2-nb with an expected size of 17 kDa, protein ladder.</i></figcaption>
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The presence of the sfGFP protein at the expected molecular weight was visible in lane 4 (Figure 3). This result confirms the successful production of GFP protein in PURE system under non-repressed conditions.
Inhibition of transcription of the sfgfp gene by the DhdR repressor
The aim of the experiments was to establish that the binding of the repressor DhdR to its operator site, dhdO, effectively inhibits transcription of a gene of interest regulated by dhdO. Then, we wanted to show that the presence of 2-HG leads to the de-repression of that gene in PURE system.
Inhibition was tested on the sfgfp reporter gene by fluorescence measurements. To determine the minimal concentration of DhdR required to obtain strong repression, sfGFP was synthesized in the presence of different concentrations of DhdR. The biochemical network model predicted a range of DhdR concentrations expected to lead to different sfGFP levels, which we experimentally tested.
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<figcaption class="normal">Figure 5: Effect of different concentrations of DhdR on the expression of a fluorescent reporter gene. Experiments 1 and 2 were performed with the same batch of PCR product from clone 8, while a new batch of PCR product from the same clone was used in experiment 3. PUREfrex2.0 was used in all conditions. The intensity value of sfGFP without DhdR was used for normalization. Excitation and emission wavelengths were 488 nm and 510 nm, respectively.
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As expected, the higher the concentration of DhdR, the stronger the repression in all three experiments (Figure 5). With the new batch of linear DNA, repression was consistently stronger. We deduced from these results that the optimal concentration of DhdR to efficiently repress expression of a gene under transcriptional control of a dhdO operator sequence was 1.5 µM, validating the predictions of the biochemical network model.
Inducible expression by 2-HG
Induction of gene expression that was repressed by 1.5 µM of DhdR was then assayed using physiological concentrations of 2-HG found around tumor cells, i.e., between 10 and 100 µM. A higher concentration was also tested, corresponding to full saturation of the DhdR repressor. The results demonstrate that 2-HG de-represses transcription of DhdR-bound DNA in a concentration-dependent manner (Figure 6). Up to 48% of sfGFP signal was recovered at a saturating concentration of 2-HG. The reason why protein production is not fully restored remains to be investigated.
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<figcaption class="normal">Figure 6: Effect of different concentrations of 2-HG on DhdR repression. PUREfrex2.1 was used. The intensity value of sfGFP without DhdR and with 10 µM 2-HG was used for normalization. Each concentration was corrected taking into account the inactivation effect of 2-HG on PURE system.
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