you can get a good quantitative summary from van urk and uv
at 530 nm

you can just add it after sterilising... They do it in a few references with heat sensitive materials that need to be introduced to the mix...

Add it to sterile water... that supplement was probably made in just about the most sterile conditions you could hope for, really Vesp I promise you don't have to worry about contams as much as you think you do.  Look up commercial procedures for mushroom farming liquid innoculants.  Not 100% sterile, you will see unstoppered liquid culture flasks everywhere.. Try a test, make a liquid culture conducive to claviceps, make 7 jars or so in a pressure cooker, to each one expose to increasing levels of contams, pour tap water in, breathe on it, pour it around... I bet you will be surprised at unfavourable the solution is for most airborn spores/etc, not to mention when there is a culture already established with its own defence mechanisms and secondary metabolites..

If one were to isolate a half decent strain first, that could solve the fluorescence issue with curcumin. Shouldn't bee a need to van-urk it, if one already knows one has a decent yield. 500mg/liter wouldn't be piss poor, 1g/l is definately within the realms of less than a few thousand hours of active lab work, as opposed to running 20 thousand plates of X number of mutagen-spiked colonies and fermenting each one until one gets to 4-5g/l without custom additions to the bog standard 8-salt broth.

And if one adds lysine+biotin, and observes yield on top of that, it shouldn't be hard to see if one gets a huge increase in yields by the EGCG/cucurmin. As for heat stability, 50mg/liter isn't too large to run through a series of filters. Pelting it with X-rays if one were able to rig up or lay hold of an X-ray tube of course could be used to sterilise a solution, and just add it without using sterifiltration techniques, and add it, bacteria and all, can't contaminate a culture with dead bugs, afterall.

Perchance not, come to think of it....its possible, if the fluorescence is a different color, that it may well enhance contrast, I have a paper somewhere where fluorescein was used for colorimetric analysis using UV, and the different coloring of the two fluorescences served as an effective contrast agent.

So if this EGCG/curcumin tricks works to activate the genes then the nutrient solution currently in literature is obsolete.  I bet a totally different mixture would be optimal.  What would be the limiting factor if this worked?  Would excessive aeration still be necessary or is that gene not affected in the same way?

There are genetic factors which regulate secondary metabolite production, indeed one way production of the good stuff has been increased, has been reversion of auxotrophic mutants deficient in their ability to produce one or more aminoacids used in the various enzymatic production stages of the ergoline ring (whilst supplementing the same to keep the fungus living and well nourished) back to a normal heterotrophic state, this apparently decouples the production of the secondary metabolites desired from the factors acting as repressors or rate-limiting steps, for example, enabling overproduction/utilization of tryptophan and making it more available for subsequent prenylation, etc.


Not sure if this holds true for ALL auxotrophs, or all aminoacids used for building the ergot alkaloid backbone, when more pressing projects (for instance the AMPAkine to hopefully unfuck my mnemonic capacity) are done and I have enough money to start work in earnest, I shall have to investigate that, if I can pull that particular mutation off in practise, I.E get lucky....hello Igor work, here I come!

Afterthought-It would be nice to know the gene sequences for those repressor genes and their function, just imagine what could come of introducing a siRNA or antisense knockdown via electroporation, viral vector, calcium phosphate technique etc, assuming those genes don't also perform some sort of process vital  for vegetative growth.

A plasmid encoding such a silencing RNA fragment or antisense oligo, should, once actually prepared, as genetic manipulations go, not be horrendously complicated (although construction of a viral vector and purification thereof could be pretty tricky if that method of transfection was chosen, calcium phosphate precipitation looks easy as hell to ghetto up, a gene gun perhaps, hell the original gene gun, if I remember properly, was hacked together out of an air pistol! Electroporation, using electric pulses to transiently punch a load of tiny holes into cells, through which such a plasmid vector could just slip in and make itself home, that could be doable in a hobbyist setting methinks.

Once in there, one's custom plasmid  would continue to produce the antisense knockdown nucleotide sequence complimentary to the sense strand coding for said repressor enzymes, and replicate within the fungus during its continued culture and propagation.

Fuck me sideways with a dead badger stuffed full of rusty nails up my arse thogh, if I could confidently predict the result on a plasmid insert after slapping it about with a whole bunch of nasty mutagens, if it were done, best done by transfecting an already decently yielding strain that one had bred already 'here's one I cooked earlier'


Think, in concept, Claviceps spp. on steroids

Not sure if its stereospecific, it may not be.

Tryptophan acts to increase yield by two mechanisms, one is simply providing more substrate for the prenylation with dimethylallyl pyrophosphate first step in constructing the ergoloid backbone, that one is the obvious one, but it also induces the transcription of the gene coding the enzyme for doing so (I think its DMAP synthetase, but it might be the gene for the next enzyme/reaction along the chain) I can't remember which, I need to find my folder with all my journal printouts on it.

That is, if the pigs didn't steal it, like they stole my good computer and a set of glassware I had to replace for forensic examination, after my perditionslut former housemate decided to wreak vengeance on me, after she went for me, and I had to bang her out, then tell her to get the fuck out of my home, and never come back. Fucking slag made up a load of completely false stories about me to the filth, who kicked my door in, stole tons of stuff which I won't get back until forensics are done taking the piss, and taking it fucking slowly at that and find nothing (for there is nothing to find)

Worse than that, some bastard pig murdered my pets, luckily the cat was unhurt, but my spiders were all killed, every last one, maybe as many as 200 false widow slings, that I had raised myself, picked through dirt by hand to find things small enough to take as prey, all of them fucking butchered.


And it could not have been an accident, I told the filth, once I was arrested, and while still in my house,  exactly where they were, and   explained that they were  very delicate, what container they were in, and to take care to move the container, as they could easily be hurt.

Glassware and other property I will get back, hopefully not damaged, and of course can be bought, whilst I caught the mother false widow myself (and they aren't common here at all in the UK, only ever seen one other, and that was a different species to the one I had as a pet) That species isn't commonly kept at all by hobbyists so I can't just buy a new one, and even if I could , it wouldn't replace the life lost, of the  loved pet that I tended for many months, and hand-fed crickets.

Fuck I hate pigs, every time I see a pig in the streets I want so much to walk up and throw acid in his face.

paul stammets is currently doing research on all kinds of endophytes he is a world reknowned mycologist.
he flame sterilizes the plant matter and then cultures from that.
you might be able to find some patents of his to get a better idea on how to culture endophytes.

I have nothing to back this up, but I think I read somewhere, that for Claviceps spp. that chloramphenicol was the antibiotic of choice.