Multicistronic Systems
In a multicistronic system, the two parts share a single promoter, which transcribes into one mRNA (compared to transcribing into two separate mRNAs, the coupling of gene expression is tighter). Each part has its own ribosome binding site (RBS) upstream. The 16S rRNA binds to the RBS, recruiting the ribosome. The binding of the ribosome is non-directional and can freely associate with any RBS to initiate translation of the downstream open reading frame (ORF).
This allows the two genes to be expressed synchronously but at different intensities. We can easily characterize the expression of the drug by observing the expression of the reporter gene. By using RBSs of different strengths, we can make the expression of RNF114 relatively stronger and that of Crimson relatively weaker.
The RBS2 sequence is from: Ribosome Binding Sites. Before the Crimson coding sequence, RBS2 was BBa_J61107 with moderate expression intensity. The RBS1 upstream of the RNF114 coding sequence is the original RBS (MCS) in the pET-32a(+) plasmid, which has stronger expression.
Crimson Reporter Protein
Original Reference: Frontiers | A Bright, Nontoxic, and Non-aggregating red Fluorescent Protein for Long-Term Labeling of Fine Structures in Neurons
The red fluorescent protein is engineered from mNeptune2 through rational mutations.
- Excitation wavelength: 588 nm
- Emission wavelength: 617 nm
Advantages:
- High brightness: Crimson is 100% brighter than mCherry and 28% brighter than mKate2.
- Low toxicity: When expressed long-term in neurons, Crimson exhibits lower toxicity. Compared to mCherry, mKate2, and FusionRed, neurons expressing Crimson have higher survival rates.
- Non-aggregating: Crimson does not form aggregates in neurons and has low phototoxicity.
- Good pH stability.
- Slow photobleaching rate: The photobleaching rate is slower than that of traditional fluorescent proteins. During long-term imaging, Crimson can maintain its fluorescent signal for a longer period.
- Good membrane targeting: Crimson-CAAX performs better than other RFP-CAAX fusion proteins in neurons, allowing clearer labeling of membrane structures.
- Suitable for multiple imaging techniques: Crimson shows good imaging performance under one-photon widefield, one-photon confocal, and two-photon microscopy.
Linker Experiments
GALA3
Flexible linker vs. Rigid linker:
We found that due to the helical structure of GALA3, using a flexible linker to connect GALA3 can affect the original structure of RNF114. We attempted to introduce rigid linkers of different lengths to isolate the GALA3 structure from RNF114. We found that rigid linkers generally have better separation effects. Among them, the structure with a GS-linker*5 - GALA - rigid linker (EAAAK)*3 has the lowest root-mean-square deviation (RMSD) value.
GFP
Drawing on the experience with GALA3, considering that the C-terminus of RNF114 is spatially close to its active region (RING finger domain) and has a helical structure, we also used a rigid linker (EAAAK)*3 to extend the C-terminus of RNF114. To avoid affecting the spatial structure of GFP, we connected GFP with a GS-linker*2 after the rigid linker (EAAAK). According to the protein spatial structure prediction results from AlphaFold3 compared to the original RNF114 protein structure, the lowest RMSD value was obtained. The final RMSD value is 1.407, indicating that the structure of RNF114-GFP is very similar to that of the original RNF114.