Cheers from UNAM_genomics Mexico team!!!
I am Mariana, the wet lab core of UNAM_Genomics Mexico team, hope
everything is going out well with your project ;).
The reason why I am writing to you is because during the last couple of
weeks we have been facing many difficulties both with our firefly (P.
pyralis) luciferases and with our luxAB from Vibrio fischeri. We have been
following your lab notebook, noticing that you are making some interesting
progress in both parts.
The problem with the P. pyralis is basically the protocols, they seem to
have few or none emission with the previous assays that we have been using
with the kind help of Edimburgh's team). On the other hand, we have been
trying to extract the luxAB from V. fischeri MJ11 genomic DNA, but E. coli
(DH5alfa) is rejecting the inserted luxAB genes. We have also been unable
to make V. fischeri glow under induction with AHL.
We were wondering whether you could help us by sharing your protocols,
references, or particular experiences about the problems we are mentioning
in this mail. Any help will be very valuable for us. We have synthesize
lumazine from P. phosphoreum and Lux Y (YFP) from V. fischeri, but as our
luxAB genes don't work, we have been unable to probe them. If you are
interested we can share them with you.
In the same way, be absolutely sure that if there is anything in which we
can help you, we will be more than happy.
Wish you the best,
Mariana Ruiz Velasco Leyva
Undergraduate Program on Genomic Sciences
Answer from Theo
Thanks for the good wishes. Our project is progressing OK, we've only got a couple of weeks yet so we're rushing to finish but we've had some nice results.
I'm sorry that you have been having problems with the wet work.
A few thoughts:
Just to check, are you adding the appropriate substrates? P. pyralis luciferase will not glow without added luciferin, for example. And if you are only using luxAB I imagine you will need to add tetradecanal. luxCDE are needed to produce this substrate.
Also, how are you detecting the light? If you can get your hands on an SLR camera, then a very long exposure with a high ISO speed works quite well. It lets you see the colour. One good source of DNA for lux light emission is James Slock we can send you some of his plasmids if you like. DH5 alpha should not be rejecting your DNA, my guess would be that you did not actually manage to get it into a plasmid which we have had problems with in the past.
We wouldn't particularly recommend lumazine, our theory was that since it came from P. phosphoreum it might not work well with the V. fischeri system, and also we found the Edinburgh BioBrick was not as described (it produced RFP.)
Our best results have come from expressing luxCDABE in E. coli, which produces light without any addition of substrate.
Let us know if we can be of any further help or if you'd like more info on our answers. Look forward to seeing you in November!
Best of luck
(and the rest of the team)
Answer from Peter
We've extracted the luxCDABE genes from James Slock's plasmids and put them under an inducible promoter repressed by araC (part BBa_I0500) this repression is relieved by adding arabinose, making the construct inducible. That seems to work reasonably well and we got some nice light output.
If you want we could send a sample to you, along with some arabinose, if you need it. I am not quite sure what the international regulations are on sending material like this.
In working with the registry P. pyralis luciferase, we found it to be a bit capricious. Apparently the luciferin uptake is pH dependent and works best around pH=5, which the cells don't really like. We tried it under the registry tetR repressed expression plasmid, which suddenly showed most activity in stationary phase, but that might be an uptake thing as well. We cloned the registry part into a new plasmid with a promoter and had it sequences and what we got turned out to be completely different from the expected sequence. We were wondering whether it could be something about the "non-standard suffix and prefix" that the description mentions, that messed up our assembly. We have now got re-synthesized, optimized versions of P. pyralis and L. cruciata luciferases and get a nice red light upon addition of luciferin. In general, firefly luciferases have some issues in living cells (most reporter assays lyse the cells first) and are significantly less bright than the lux system. What promoter system and what luciferin concentration are you using?
We were able to see the light in a darkened room and took photos with different cameras at high ISO (3200 or 6400), 30s exposure with a large aperture. the cells looked quite bright under those conditions, but only if all stray light was eliminated.
We use an automatic plate reader for most of our measurements. while not providing pretty pictures, it gives more quantifiable results and allows time-course readings.
(and the rest of the team, again)