Our reporter system consists of two key components: our modified pcDNA5 plasmid and our reporter plasmid. Using these two components, we aim to dynamically measure the levels of compound D-2HG within our system by utilizing the transcriptional repression factor DhdR, a protein that has been identified in A. denitrificans. We constructed and utilized a variety of basic and composite parts in the build-up of our system. For more information about our reporter system design visit our Engineering page.
As part of our constitutive reporters, we used the mCherry gene, which allows us to dynamically measure the levels of the compound of interest in our system. In addition, we used several previously documented pieces, CMV promoter (BBa_K1119006), bghA terminator (BBa_K1150012), and mCherry (BBa_K180008).
To construct our IDH1-specific reporter system, we utilized a newly identified transcription repression factor, DhdR (Xiao et al, 2021). This protein was initially isolated from A. denitrificans, and was identified as being sensitive to D-2HG levels within the cell. Since the DhdR protein is a transcription factor, it has to be localized to the nucleus after expression. Therefore, we added a nuclear localization sequence (NLS) to the construct. In addition, a FLAG tag was attached for ease of Western blotting to validate that the protein was being expressed in mammalian cells. To account for possible effects these additions could have on protein folding, we created two constructs: DhdR-NLS-FLAG and FLAG-NLS-DhdR. Implementing these two variations allows us to test which sequence order leads to optimal expression. During our design process, we also used a human-codon optimized version of the DhdR sequence because our system is modeling human glioma.
We determined the two best dhdO binding sites based on DhdR’s binding affinity to each sequence, and constructed our reporter plasmids using a variation of these two sites.
Through literature research, we also identified that the inclusion of spacer sequences (GTCGAG) may alter the binding behavior which motivated us to test several variations of the two binding sites with the inclusion of the spacer sequence [1]. Additionally, we were interested in determining if cooperativity played a role in binding affinity, so we created additional constructs with multiple repeats on the same binding site sequence, as well as versions that included the spacer sequence to determine if there were steric interactions at play [2].
We also wanted to establish an IDH1 positive cell line for use in our co-culture system. To achieve that, we inserted coding sequences of wild type IDH1 and IDH1 R132 mutation into pcDNA5 plasmid.
The table below lists the new parts that Duke iGEM has contributed:
| Part Name | Description |
|---|---|
| K4046050 | Kozak Sequence (Human RBS) |
| K4046010 | DhdR-NLS-FLAG (bacterial repression factor) |
| K4046000 | FLAG-NLS-DhdR (bacterial repression factor) |
| K4046100 | DhdO binding site #1 |
| K4046200 | DhdO binding site #2 |
| K4046300 | DhdO binding site #1 with spacer |
| K4046400 | DhdO binding site #2 with spacer |
| K4234000 | IDH1 R132 coding sequence (Homo sapiens) |
| K4234001 | IDH1 WT coding sequence (Homo sapiens) |
As part of our final goal, we intend to construct a library of composite parts, which consist of different combinations of the binding site sequences and spacers. Several of these parts have been constructed, and establishment of the remaining composite parts is ongoing. After all composite parts have been constructed, we intend to test the various combinations for efficacy in our system.
The table below lists the composite parts that Duke iGEM has built or intends to build using CMV as a promoter:
| Part Name | Description | Part Name | Description |
|---|---|---|---|
| K4046510 | CMV-Kozak-cLuc | K4046830 | CMV - BS #1 - BS #1 - BS #1 - Kozak - cLuc - bghA |
| K4046520 | CMV-Kozak-cLuc | K4046840 | CMV - BS #1 with spacer - BS #1 with spacer - BS #1 - Kozak - cLuc - bghA |
| K4046610 | hUBC-Kozak-tdTomato | K4046850 | CMV - BS #2 - Kozak - cLuc - bghA |
| K4046620 | hUBC-Kozak-cLuc | K4046860 | CMV - BS #2 - BS #2 - Kozak - cLuc - bghA |
| K4046700 | CMV - BS #1 - Kozak - tdTomato - BghA | K4046870 | CMV - BS #2 with spacer - BS #2 - Kozak - cLuc - bghA |
| K4046710 | CMV - BS #1 - BS #1 - Kozak - tdTomato - BghA | K4046880 | CMV - BS #2 - BS #2 - BS #2 - Kozak - cLuc - bghA |
| K4046720 | CMV - BS #1 with spacer - BS #1 - Kozak - tdTomato - BghA | K4046890 | CMV - BS #2 with spacer - BS #2 with spacer - BS #2 - Kozak - cLuc - bghA |
| K4046730 | CMV - BS #1 - BS #1 - BS #1 - Kozak - tdTomato - BghA | K4046900 | CMV - BS #1 - Kozak - mCherry - bghA |
| K4046740 | CMV - BS #1 with spacer - BS #1 with spacer - BS#1 - Kozak - tdTomato - BghA | K4046910 | CMV - BS #1 - BS #1 - Kozak - mCherry - bghA |
| K4046750 | CMV - BS #2 - Kozak - tdTomato - BghA | K4046920 | CMV - BS #1 with spacer - BS #1 - Kozak - mCherry - bghA |
| K4046760 | CMV - BS #2 - BS #2 - Kozak - tdTomato - BghA | K4046930 | CMV - BS #1 - BS #1 - BS #1 - Kozak - mCherry - bghA |
| K4046770 | CMV - BS #2 with spacer - BS #2 - Kozak - tdTomato - bghA | K4046940 | CMV - BS #1 with spacer - BS #1 with spacer - BS #1 - Kozak - mCherry - bghA |
| K4046780 | CMV - BS #2 - BS #2 - BS #2 - Kozak - tdTomato - bghA | K4046950 | CMV - BS #2 - Kozak - mCherry - bghA |
| K4046790 | CMV - BS #2 with spacer - BS #2 with spacer - BS #2 - Kozak - tdTomato - bghA | K4046960 | CMV - BS #2 - BS #2 - Kozak - mCherry - bghA |
| K4046800 | CMV - BS #1 - Kozak - cLuc - bghA | K4046970 | CMV - BS #2 with spacer - BS #2 - Kozak - mCherry - bghA |
| K4046810 | CMV - BS #1 - BS #1 - Kozak - cLuc - bghA | K4046980 | CMV - BS #2 - BS #2 - BS #2 - Kozak - mCherry - bghA |
| K4046820 | CMV - BS #1 with spacer - BS #1 - Kozak - cLuc - bghA | K4046990 | CMV - BS #2 with spacer - BS #2 with spacer - BS #2 - Kozak - mCherry - bghA |
| K4234300 | CMV-IDH1-Kozak-bghA | K4234400 | CMV-IDH2-Kozak-bghA |