Green FPs in HEK293T cells

At one point, I was a doe-eyed postdoc reading about new fluorescent proteins (FPs) with improved brightness and thinking it was potentially important to incorporate new FPs into my constructs for cell culture work. I have since come to realize that the intrinsic gains to fluorescence published in those papers do not necessarily translate to brightness in my experiments. The reason are probably multifactorial, including:
1. Commonly accessible equipment is generally optimized for EGFP (or similar FPs), so newer FPs with slightly different excitation and emission spectra may not be captured well with existing microscopes or flow cytometers.
2. The FP brightnesses are typically assess in-vitro, and there may be other factors in eukaryotic cell cytoplasms that may affect the FP brightness (eg. FP half-life).

Well, I’ve still ordered a handful of different green fluorescent proteins anyway, and figured it was worth doing a side-by-side comparison in the transgenic system we use in the lab. This was all done with the HEK293T G542Ac3 (LLP-Tet-Bxb1attP_Int-BFP_IRES-iCasp9-BlastR) cells, which were stably recombined with a single copy of each fluorescent protein at a common genomic locus. The construct organization was: Bxb1attB_[Green FP]_IRES-mCherry-2A-PuroR. Olivia did these recombinations, selected the cells, and ran the cells on the ThermoFisher Attune Flow cytometer, with Sarah’s help. This is what the results look like:

Some interpretations:
1. Rather minimal (~3-fold) difference between mGreenLantern and UnaG. I suppose if we were ever in a situation where we needed every unit of green brightness possible, that we would go with mGreenLantern. That said, UnaG has some benefits; namely, it’s 58% the size of EGFP/mGreenLantern/mNeonGreen, and it lacks the VERY ANNOYING identical sequences at the N- and C-termini (MVSKG … DELYK) which makes molecular cloning a potential pain.
2. Conceptually, I like the idea of fuGFP, but that 20-fold diminished green fluorescence compared to EGFP is potentially problematic. Who knows, maybe I’ll turn this into a target of directed evolution at some point…?