firefly luciferase regenerating enzyme

[poli]

Anyone have any info on expression of the firefly LRE in plants in combination with luciferase? The cambridge igem team performed their work in e coli and found d-cysteine was a necessary component. The glowing plant project has conflicting info on which luciferase system they are using and I haven't seen any publications except Krichevsky et al 2010 Plos One (who used vibrio). Anyone know if coexpression of LRE with luciferase has been shown to regenerate any luciferin without addition of d-cysteine?

[Mega [Andreas Stuermer]]

It's just luciferin *regenerating* enzyme, not producing. Thus you still have to add luciferin.

 

AFAIK, they have done in the US both firefly luciferase (probably plus LRE) for a photo and the main work is how to implement vibrio fischeri lux.

[poli]

I have luciferase expressed in my algae which I feed luciferin (this works well), I would like to work with the regenerating enzyme if there is some indication it works under normal cell conditions to *regenerate* the spent luciferin (and to further test my system). Luciferase timecourses show a diminished signal over a time which I would like to combat. The igem team seemed to find d-cysteine is necessary to regenerate the correct chiral luciferin and I want to know from if this is necessary in vivo in a eukaryote. Some claim d-cysteine is present in the peroxisome and the luciferase + LRE tend to localize there, or luciferase can change the chirality of l-luciferase to d-luciferase. I could express the LRE with luciferase in the algae with d-cysteine and chbt in the media and screen for luciferase signal to see if it works but I don't want to spend $300 and a month of work before feeling out what does and doesn't work for others. Anyone express LRE and show it works, if so under what conditions?