Synthetic uORF construct

So I’ve long wanted finer control of protein abundance, and to date, have had the greatest success messing with the Kozak sequence to alter translation rate, thus modulating the amount of protein steady-state abundance. That said, I’ve wondered if there are other aspects that could be further manipulated to increase the dynamic range of the amount of protein steady-state abundance. This at some point led me to try playing around with upstream open reading frames (uORFs), that can interfere with the translation rate of the downstream protein (in my case, a green fluorescent reporter). We recently made a vector with one such uORF, so I looked at what effect having that uORF had on green fluorescence of the cells.

The actual vector plasmid name is “AttB_2xuORF_mGreenLantern-T2A-shBle-IRES-mCherry-P2A-PuroR”. As you can tell, red fluorescence is behind an IRES, and should be unaffected by the uORF. That’s indeed what we see, with the red distribution being a control construct without the uORF, and the blue distribution being the identical construct with a uORF immediately preceding mGreenLantern (Note: YL2-A is the fluorescence channel for red fluorescent emission).

Now if I gate on that bright red population, and look at the amount of green fluorscence, there is indeed a difference (BL1-A is the channel to look at for green fluorscence), although the effect isn’t huge. Looking at the distribution geometric means, it looks like the uORF construct is roughly 3.23-fold less bright than the control, so roughly a half-log less bright. Now the reason I’m underwhelmed is that I can get a roughly 1.5-fold difference in fluorescence using different Kozak sequences, so the uORF that I created doesn’t exhibit nearly the same magnitude of effect. Eh, that’s designing and testing constructs for you. That said, I suppose if I were to combine both, then I could likely get > 2 logs of dynamic range.

PS. Yes, I know there are simpler ways to modulate protein abundance, like transcriptionally through modulating the amount of expression. One day we’ll do more of that, but for now, translational control it’s been.