Re: Can one plasmid code for both firefly luciferase and luciferin?

Dear Scott,

Of course, I could just give you a laconic answer (“no, you can’t”) but I 
suspect that you want to understand the reasoning behind it!

The luciferase reaction is catalysed by a protein (luciferase): the 
organic compound luciferin (or 3,7-dihydro-2-(p-hydroxybenzyl)-6-(p-
hydroxyphenyl)-8- benzylimidazo[1,2-a]pyrazin-3-one :-) reacts with 
oxygen and ATP to produce oxidised luciferin, pyrophosphate, AMP and 
light. So you see, you can express the protein, but in order to “express” 
luciferin in a system that does not already contain it, you would need to 
express all the enzymes involved in the synthesis of this compound from a 
precursor. I must admit that I don’t know what steps are needed: I tried 
to find some information on the internet but it would seem that the 
closest one has come is to acknowledge that the dear bug uses cystein 
somehow! :-)

The green fluorescence protein (GFP) is also a one protein system: two 
amino-acids within its structure cyclise to form a so-called conjugated 
pi-electron system that can fluoresce. You do not need to add anything 
and so you can express the one protein in a cell and get fluorescence. 
But please note that this is fluorescence and not luminescence: you shine 
light of a certain wave-length at it: it will absorb this and emit light 
at another wave-length. This requires no energy input, except of course 
for the actual synthesis of the protein!

Vibrio fischeri is a bacterium that lives freely in sea water. It can 
colonise special organs of a small squid (Euprymna scolopes) and when 
this happens, it can be triggered to emit light. It also associates with 
some fish. As opposed to the fluorescence of GFP, the luminescence of V. 
fischeri involves the oxidation of a compound (an aliphatic aldehyde) 
coupled to the emission of light, hence the need for energy, just as with 
our fire-fly luciferase. This explains why it will not glow while living 
freely, but only when confined to the energy-rich environment of its 
host’s light-emitting organ. On the other hand, you do not need to add an 
odd compound such as luciferin to get light emission: many endogenous 
aliphatic aldehydes will do.

So, GFP is not a luminescent protein: it picks up light and emits light. 
This is fluorescence and an “uncomplicated” process insofar as it does 
not require anything else than itself to be seen. Luciferase from fire-
fly (Luc) and from the coral sea pansy (Ren) can be expressed from a 
single gene and will use luciferase or coelenterazine as substrates, 
respectively. The enzymes needed for bacterial luminescence in (for 
instance!) V. fischeri is coded by a group of genes (the lux operon, 
which makes it a more complex system) but does not require an odd 
compound and hence you only need to express the protein(s) to get light.

I would suggest that, for the purpose of your course, you either explain 
the chemistry of the fire-fly luminescence, as this is a known 
phenomenon, or use the V. fischeri system as this requires no odd 
additions. For Luc, maybe say that you need a chemical switch (the fly 
lights up in patterns, group-specific). For the lux operon, you might 
find a natural science film showing how the bobtail squid flashes it’s 
light to hide its shadow.

Although I couldn’t answer your request for a “simple fix” I hope that 
you have some use of my rather lengthy negative answer!

Kind regards,

Erik