To create a robust FRET measurement system, we first needed a simple staple to bring two DNA strands into proximity
and
identify suitable FRET pairs for efficient measurement. Our design process began with extensive literature research
to
select well-characterized DNA-binding proteins and fluorescent pairs that were proven to work in E. coli without
compromising binding efficiency when fused. Among the potential candidates—Oct1-DBD, Gal4-DBD, tetR, and
NFKB-DBD—Oct1-DBD was the only naturally monomeric protein. We hypothesized that ths monomeric nature is important
to
ensure stoichiometric binding at staple target sites.
We selected Oct1-DBD as one DNA-binding protein due to its proven strong expression and binding affinity in E.
coli. Our
second protein was an engineered version of the well characterized Tetracycline Repressor (tetR) that forms a
functional
monomer. This scTetR is a fusion of two tetR proteins with a flexible linker which was described to maintain the
same
DNA-binding affinity and specificity as wild-type tetR.
For the FRET pairs, we established the following key criteria: small size, monomeric, minimal photobleaching, high
fluorescence and quantum yield, and emission outside the blue and near-infrared spectra to reduce cell damage and
background fluorescence. After reviewing literature and using FPbase to predict FRET efficiency, we chose mNeonGreen
and mScarlet-I for their superior performance in FRET applications both reported in literature and calculated with
FPbase.
We constructed a two-plasmid system: an expression plasmid and a folding plasmid. The expression plasmid contained the following required proteins: scTetR-Oct1 fusion (staple), scTetR-mScarlet-I (FRET acceptor), and Oct1-mNeonGreen (FRET donor), additionally the vector had 15 repeats of the tet response element (TRE) to ensure efficient tetR binding. The folding plasmid was designed with 15 repeats of the Oct1 binding motif. To ensure compatibility, special consideration went into deciding the origin of replications (ori), we selected a high-copy pMB1 origin for the expression plasmid and a medium-copy p15A origin for the folding plasmid.
To maintain balanced expression and minimize metabolic burden, we designed a polycistronic expression cassette under the T7 promoter. This allowed the transcription of one mRNA encoding all three proteins, each with its own ribosome binding site (BBa_B0030). To facilitate future modifications, we included restriction sites between the ORFs and at strategic locations. Gene synthesis was used to generate the inserts, which were cloned into the backbones using Gibson assembly.
As controls, we constructed a positive control by fusing mScarlet-I and mNeonGreen to observe direct FRET efficiency, and a negative control by using a folding plasmid lacking the Oct1 target sequence.
Fluorescence intensity for mNeonGreen, mScarlet-I, and FRET was measured 18 hours post-incubation with varying IPTG concentrations (0.8 mM to 0.0125 mM).
Figure 1: Fluorescence measurement of first simple staple construct
Fluorescent measurement, normalized to cell count, of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560
nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) in E. coli, 18 h
after induction with different IPTG concentrations. Data is presented as mean ± SD.
The positive control exhibited strong fluorescence for both proteins and FRET, indicating that the system was
functional in principle. However, the constructs expressing the staple and FRET-pairs didn’t exhibit any
fluorescence for all three measurements, indicating an issue with protein expression
The lack of fluorescence in the polycistronic constructs suggested problems in transcription or translation possibly due to the metabolic burden of expressing large, complex constructs. This hypothesis is also supported by the positive control where increasing fluorescence could be measured with decreasing IPTG concentrations, reinforcing the idea that overexpression might be causing stress and inhibiting efficient protein production. Importantly this initial testing showed that the FRET pairs are compatible resulting in measurable fluorescence readout, motivating us to further pursue this line of experiments.
