Overview

Our reporter system consists of two connected pieces. We aim to dynamically measure the levels of compound D-2-HG within our system by utilizing the transcriptional repression factor DhdR, a protein that has been identified in A. denitrificans. As such, we have constructed and utilized a variety of basic and composite parts in the build-up of our system.

Basic Parts

As part of our constitutive reporters, we use the cLuc gene and the tdTomato gene, which allow us to dynamically measure the levels of the compound of interest in our system. In addition, we used several previously documented pieces, including hUBC promoter (BBa_K2557034), CMV promoter (BBa_K1119006), bghA terminator (BBa_K1150012) and mCherry (BBa_K180008).

In order 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-2-HG levels within the cell. Because the protein awas a transcription factory, it had to be localized to the nucleus after expression. This necessitated the attachment of a nuclear localization sequence to the construct. In addition, a FLAG tag was attached for ease of Western blotting. In order to account for possible effects on protein folding, we utilized two different constructs, one where the additions are made at the end and one where the additions were made at the start. This should allow us to test for which sequence order leads to optimal expression. Also, the DhdR sequence used was human-codon optimized to facilitate expression in our system.

The two DhdO binding sites selected for usage were chosen because they were identified as having high binding affinity to the DhdR repressor protein. Through literature research, we identified that the inclusion of spacer sequences may alter the binding behavior, so we also tested versions of the two binding sites that had spacer sequences attached.

The table below lists the new parts that Duke iGEM has contributed:

Table 1. Basic parts

Part Name	Description
K4046050	Kozak Sequence (Human RBS)
K4046500	cLuc (Cypridina luciferase)
K4046600	tdTomato (fluorescence protein)
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

Composite Parts

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:

Table 2. Composite parts - CMV

Part Name	Description	Part Name	Description
K4046510	CMV-Kozak-tdTomato	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

The table below lists the composite parts that Duke iGEM has built or intends to build using huBC as a promoter:

Table 3. Composite parts - huBC