← Library
David W. Ow, Keith V. Wood, Marlene DeLuca, Jeffrey R. De Wet, Donald R. Helinski, Stephen H. Howell
Science, New Series, Vol. 234, No. 4778 (Nov. 14, 1986), pp. 856–859
- ATP isn't the bottleneck. Plant cells have enough endogenous ATP to drive the luciferase reaction without adding any externally. Plant cells will have plenty of ATP for firefly luciferase.
- You need very little enzyme to get a signal. 300–900 luciferase molecules per cell gave detectable light on a luminometer. With a strong promoter you'll have orders of magnitude more protein than that. The enzyme side of the equation is easy, but the substrate supply is the entire problem.
- Terminator choice matters. Deleting the firefly's own polyA signal crushed expression to 8%. In your multi-gene Golden Gate constructs, don't treat terminator selection as an afterthought for any of your four cassettes. Each one affects mRNA stability and protein levels.
- 35S promoter is brightest in roots. They saw higher expression in roots than leaves, confirmed independently. For proof-of-concept agroinfiltration in N. benthamiana leaves this doesn't matter. But if you ever go to stable transgenics, consider that 35S might make your plant glow brightest underground. Leaf-specific promoters might be worth testing later.
- Exogenous luciferin delivery is terrible. They needed 1 mM luciferin in 20% DMSO, 6 hours of root watering, and a 24-hour film exposure. The signal was a mess of transport artifacts, not biology.
- Exogenous luciferin is also toxic. Kills cells above 400 µM. The enzymatic pathway should produce luciferin at low steady-state concentrations, continuously replenished but never hitting toxic bolus levels.
- The sensitivity advantage is real. They detected 3×10⁶ luciferase molecules and claimed 100× better sensitivity than CAT assays. Even modest endogenous luciferin production combined with efficient luciferase should produce a detectable signal.
- Bottom line from this paper: The enzyme works great in plants. Always has, since 1986. The only thing that's ever been missing is the substrate.