Overview
Goal:
As part of NODES, we created a genetic reporter system that can dynamically change based on the levels of D-2-HG in IDH1-mutated cells. Our genetic reporter system consists of two parts: 1) a plasmid to express the allosteric transcription repressor DhdR and 2) a constitutive reporter plasmid containing binding sites for DhDR. When D-2-HG levels are elevated in glioma cells due to the IDH1 mutation, D-2-HG interacts with DhdR and releases it from the binding site on the constitutive reporter plasmid, allowing for proper transcription of the downstream reporter protein sequence.
Results:
We designed four fluorescent and luminescent constitutive reporters plasmids and verified their gene expression in HEK 293T cells using fluorescence microscopy and a luminescence assay. We also built an initial line of binding site plasmids for use in our genetic reporter system and tested them for baseline impacts on fluorescence expression. Finally, we constructed a Sanger-sequencing-verified version of the plasmid that encodes our allosteric transcription factor DhdR.
Future Plans:
During Phase 2 of our project, we will determine the most optimal version of the binding site, promoter, and reporter gene plasmid that provides the best working range of fluorescence or luminescence, which will allow for visualization of relevant changes in D-2-HG. We will fit our experimental data into our computational model to inform which reporter variation best encompases glioma-relevant ranges of D-2-HG. To verify protein expression of DhdR, we will run a Western blot. We will then co-transfect the binding site plasmid with the DhdR protein plasmid in HEK293T cells and later BXM primary glioma cells to test our proposed reporter system. Finally, we will culture our reporter-transfected glioma cells with our brain organoids to establish the final testing platform.
Fluorescent and Luminescent Constitutive Reporters
To develop a high-throughput drug screening platform, we needed to determine methods of characterizing cell behavior without lysing our samples. We first selected reporter genes (tdTomato and cLuc) that encode fluorescent and luminescent proteins, respectively, that have the ability to induce visually identifiable responses. To establish the backbones for creating our overall reporter system constructs, the team designed four plasmids consisting of different promoter (CMV and hUBC) and reporter gene (tdTomato and cLuc) combinations (Figure 1).
Figure 1. Plasmid maps of the Constitutive Reporter constructs. Top Left: CMV with tdTomato. Top Right: CMW with cLuc. Bottom Left: hUBC with tdTomato. Bottom right: hUBC with cLuc
Figure 2. Gel Electrophoresis Verification of Constitutive Reporter PCR fragments, Ladder: 2-Log DNA Ladder, Lane 1: huBC, Lane 2: huBC, Lane 3: cLuc, Lane 4: tdTomato, Lane 5: CMV, and Ladder: 2-Log DNA Ladder
After joining the purified PCR fragments using Gibson Assembly (Figure 2), we verified all four of our cloned plasmids via Sanger sequencing. For more details on the Gibson Assembly process, please consult the Experiments page. (https://2021.igem.org/Experiments.html)
Verification of tdTomato Constructs in HEK293T Cells:
To verify the fluorescence gene expression of tdTomato, we transfected huBC-tdTomato into HEK293T cells, a widely used cell line, for initial validation of our construct. The cells were successfully transfected with moderate efficiency (~40%) and demonstrated fluorescence expression (Figure 3). The successful fluorescence expression of huBC-tdTomato in HEK293T cells validates that our construct can be used as an imaging marker for synthetic biology applications to characterize different cell behaviors, such as changes in D-2-HG.
We are currently repeating this experiment to transfect both huBC-tdTomato and CMV-tdTomato in HEK293T cells. Using fluorescence microscopy and flow cytometry data, we will compare their fluorescence expression levels to determine how promoter type affects fluorescence expression.
Figure 3. Fluorescent microscopy verification of the expression of the huBC-tdTomato construct in HEK293T cells.
Verification of cLuc Constructs in HEK293T Cells:
To verify the CMV-cLuc and huBC-cLuc constructs, we transfected them into HEK293T cells and performed a luciferin-based luminescence assay. We added vargulin, a luciferin that releases light in the presence of a luciferase (Figure 4), to HEK 293T cells transfected with various amount of CMV-cLuc and hUBC-cLuc DNA (0 ng, 100 ng, 250 ng, and 500 ng).
Figure 4. Luminescence of Cypridina Luciferin in the presence of the enzyme Cypridina luciferase (cLuc) (Thermo, 2021)
The HEK293T cells with no transfections had no fluorescence intensity. The fluorescence intensity was significantly greater in cells with CMV-cLuc (Figure 5, left) and huBC-cLuc (Figure 5, right) transfected compared to non-transfected cells, verifying that the two cLuc constitutive reporters we designed secreted luciferase enzymes. The relative fluorescence enzyme intensity tends to increase as the amount of DNA transfected increases, but further testing is required to determine whether transfected cells secreted luciferase enzyme into the media in a concentration-dependent fashion.
Figure 5. Luciferase Assay of CMV-cLuc (left) and huBC-cLuc (right) in HEK293T cells with various DNA transfection amounts (0 ng, 100 ng, 250 ng, and 500 ng)
Allosteric Transcription Factor DhdR Plasmid
Based on our proposed system design, we constructed a plasmid to express the allosteric transcription repressor DhdR. We have two versions of the construct with different nuclear localization sequence and FLAG tag placements (DhdR-NLS-FLAG vs. FLAG-NLS-DhdR) and will clone them into pcDNA5 plasmids to determine which sequence order allows for better protein expression (Figure 6).
Figure 6. Plasmid maps of the DhdR constructs. Left: CMV with DhdR-NLS-FLAG. Right: CMW with FLAG-NLS-DhDR.
We used Gibson Assembly to join together the purified PCR fragments (Figure 7) and construct CMV-DhdR-NLS-FLAG and CMV-FLAG-NLS-DhdR plasmids. We have successfully verified the DhdR-NLS-FLAG plasmid via Sanger sequencing (Figure 8) and are currently in the process of cloning the FLAG-NLS-DhdR plasmid.
Figure 7. Gel Electrophoresis Verification of DhdR PCR fragments, Ladder: 2-Log DNA Ladder, Lane 1: DhdR-FLAG, Lane 2: FLAG-DhdR, Lane 3: pcDNA5, Lane 4: pcDNA5, and Ladder: 2-Log DNA Ladder
Figure 8. Comparison of the Sanger sequencing results of our constructed CMV-FLAG-NLS-DhdR with the expected sequence on Benchling.
Verification in HEK Cells:
To verify the protein expression, we transfected the DhdR-NLS-FLAG plasmid into HEK293T cells. We are currently in the process of running a western blot to detect and analyze the presence of DhdR. After the CMV-FLAG-NLS-DhdR plasmid is built, we will repeat this process to determine the sequence that yields higher protein expression.
DhdR Binding Site Constructs
To construct the second component of our reporter system (the DhdR binding site constructs), we inserted ten variations of the binding site constructs into a commercially available pcDNA5 plasmid, which encodes for the red fluorescent protein mCherry. Five of the ten constructs have been successfully ligated together and verified via Sanger sequencing (Table 1, Figure 9).
Table 1. Sanger Verification of Binding Site Constructs Inserted in pcDNA5
|
Plasmid Label |
Binding Site Constructs Inserted in pcDNA5 |
Sanger Verified? |
|
BS 1 |
dhdO 0# |
Yes |
|
BS 2 |
dhdO 5# |
Yes |
|
BS 3 |
dhdO 0# (2 Repeats) |
- |
|
BS 4 |
dhdO 0# (2 Repeats with Spacer) |
- |
|
BS 5 |
dhdO 5# (2 Repeats) |
Yes |
|
BS 6 |
dhdO 5# (2 Repeats with Spacer) |
- |
|
BS 7 |
dhdO 0# (3 Repeats) |
Yes |
|
BS 8 |
dhdO 0# (3 Repeats with Spacer) |
- |
|
BS 9 |
dhdO 5# (3 Repeats) |
- |
|
BS 10 |
dhdO 05# (3 Repeats with Spacer) |
Yes |
Figure 9. Sanger Verification of Binding Site Constructs. The figure shows the sequence information for our different constructs of varying repeats and spacers, and green check marks indicate the constructs that have been verified through Sanger sequencing.
Since we are determining relative D-2-HG levels based on fluorescence levels, it is important to ensure that no other factors change fluorescence levels besides changes in D-2-HG. We checked to see if inserting the DhDR binding sites into the pcDNA5 plasmid affected the fluorescence levels of mCherry.
Verification in HEK Cells:
To verify the fluorescence gene expression of our DhdR binding site constructs, we transfected the five Sanger verified constructs, pcDNA5 as a positive control, and an unverified BS 3 construct as a negative control into HEK293T cells.
Figure 10. 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.
From direct visual analysis, it appears that the inclusion of binding sites might affect the expression of the downstream reporter genes, even in the absence of DhdR protein (Figure 10). For instance, the level of red fluorescence in the BS 2 sample was quite low, as compared to the original pcDNA5 control. Although this could have resulted from non-ideal lipofection, it could have also been the result of direct binding site impacts on the expression of the mCherry protein. On the other hand, the other binding sites did not appear to have significant impacts on expression levels, as indicated by the relatively high levels of fluorescence. However, fluorescence imaging fails to provide a quantitative measure of the protein expression, meaning that definitive conclusions cannot be drawn from this data alone. Therefore, we are currently in the process of running flow cytometry on our samples to quantify fluorescence expression. Using this data, we will compare the fluorescence expression between the pcDNA5 control and the verified binding site constructs to assess whether the insertion of binding site sequences affects the expression of mCherry.
DhdR Binding Site Constructs with Constitutive Reporters
After successfully constructing our constitutive reporters and ligating the binding site constructs into commercially available plasmids, we are combining these two components that we have verified by inserting the binding site sequences into the fluorescent and luminescent reporter plasmids that we designed. As of now, we have constructed twenty plasmids, with ten binding site variations inserted into either the CMV-tdTomato or huBC-tdTomato reporter plasmids.
Future Work
During the phase II of our project, we will:
- Repeat the transfection of huBC-tdTomato and CMV-tdTomato in HEK293T cells to determine how promoter type affects fluorescence expression using flow cytometry and fluorescence microscopy.
- Insert the ten binding site variations into our four constitutive reporters (CMV-tdTomato, huBC-tdTomato, CMV-cLuc, and huBC-cLuc).
- Transfect our DhdR binding site and constitutive reporter plasmids into HEK 293T cells and BXM glioma cells.
- Run a western blot to verify the expression of DhdR in HEK 293T cells.
- Using luciferase assays, fluorescence microscopy and flow cytometry, and our computational model, determine the combination of the binding site, promoter, and reporter gene plasmid that provides the best working range of fluorescence or luminescence for visualization of relevant changes in D-2-HG.
References
Thermo Scientific™ Pierce™ Firefly Luciferase Glow Assay Kit, https://www.thermofisher.com/order/catalog/product/16176