FYI: our extremely capable iGEM team tried the mod GFP to make pre-coelenterazine a while back and the patent appeared not to be true in our hands (it was not usable as a substrate for Gaussia or Renilla luciferase, we used a luminometer and all the proper controls). We had asked the inventors and they told us IIRC that they had not pursued it nor proven it since then. So it appears to be a strictly theoretical patent rather than a real coelenterazine synthesis pathway and in our hands it was not a worthwhile venture. Still no papers based on this patent of mod-GFP either.
soooo... good luck!!
jordan
On Wednesday, December 26, 2012 9:21:51 AM UTC-8, Mega wrote:But there is a very big question:
The pre-czn-peptide is based on this
http://www.bio.davidson.edu/courses/molbio/restricted/02gfpseq/GFPseqfig2.GIF
Nucleotide 736-739 (in frame!) is TAA so the jellyfish polymerase will stop and fall of the mRNA. Then it goes on with ATG TCC ..... That is junk DNA? I would expect it to be an apo-protein or something alike. But jellyfish machinery doesn't support operons, so this wouldn't be expresssed.
--
No time to look it up (my wife says "no work on vacation!" and this is starting to look too much like work ;-), but make sure you're using the right genetic code - jellyfish may well use something nonstandard - and take any introns into account.
The GFP sequences we use every day have typically been recoded for E. coli, so they won't have any nonstandard codons or introns.
Patrik
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