I have uploaded a description of the lifecycle to the articles of interest in books and journals thread in the reference section.

It sounds like you have done this before... I think you may be right that random mutations are not going to be easy, but when it comes to the genetic engineering, I would need to use electrophoresis, buy probably expensive restriction enzymes and all sorts of stuff to isolate or double the wanted genes, Correct?

I have worked with these techniques a bit in the past yes but never with this organism and I'm very interested to see how any cultivation of the species goes. The enzymes could be isolated from nature depending on the sequences of interest but it would be a worthwhile intellectual exercise to investigate the process any way as it will bring you through the biosynthetic pathways of the organism and the engineering process.


There is no need for a spectrophotometer if one is willing to make serial dilutions of the sample to increase the difference in colour between the controls and the sample of interest. Once you keep the dilutions consistent you can tell how much more (or less) intense the colour is in the sample relative to the control. The only cost is time.

with
no significant preference for either of the two
mutagens or media. All high-yielding mutants
showed a violet-brown pigmentation and an in-
creased tendency toward sclerotia-like morphol-
ogy compared to the parent strain, which is
white with slender vegetative hyphae and rela-
tively low fat content

I thought this was useful -- perhaps the darker colonies will contain higher alkaloid concentration. A problem of the ergot changing color though is that it screws up the tests that I was planning to rely upon to test for the conc. of alkaloids.

I would take any such rules with a grain of salt and stick to your colour assay method. They said all high yielding mutants but few of their mutants were high yielding. Any similarities are well worth taking in your notes though.


My main concern would be the identification of the species of interest and I will take a look at the forensic literature to see if there is a non genomic identification that is easy to perform

Hey Vesp, think we should split this or rename it, so we have a pure Claviceps thread? solely dedicated to Claviceps species and the cultivation and yield-optimization thereof?

Sclerotia like morphology IS a likely indicator of a potential high yielder, as the fungus does not naturally form alkaloids unless it forms sclerotia.

Productive cycle is around 10-15 days usually for many strains, although some have longer or shorter by a bit. Immobilized mycelium is different, most un-immobilised cultures start to degrade after a few medium changes, but immobilization in alginate (2.5% is usual, but up to 7% gives much more durable beads, as does forming a layer of low molecular weight chitosan around the surface of the beads at the expense of interfering with oxygen transport and giving largely clavines, however that is without using a perfluorocarbon emulsion to form the alginate mix and supplementing the air intake with O2 hasn't been tried yet I don't think either, but I bet it works)

With immobilization, the culture can simply have medium drained off for extraction and topped up with fresh stuff, taking phosphate concentrations and likely uptake into account however and can remain stable for many cycles of production.

Other known factors which increase alkaloid output are:

Addition of biotin and leucine to the medium, according to Appl Environ Microbiol. 1983 May; 45(5): 1694–1696 Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58 gives a huge increase in productivity of around 75%

Control, grown on NL-46 medium gave 351.08 uG/ml
Addition of biotin (3 uG/ml) alone gave 615.80 uG/ml and a doubling in the number of chlamydospores present in the medium
Addition of avidin, a protein which has a scary tight binding affinity for biotin to KO biotin availability took a huge crap on yields, maybe 1/3rd of control value
Addition of leucine alone gave 409.10uG/ml
Addition of both biotin and leucine took the yield up to 712.0 uG/ml.

Which means avoid using egg white for a protein source if for any reason one was going to, as that is a good source of avidin, and it will bind and prevent use of biotin.

Increase produced by leucine alone was around 16.5%.

Phosphate restriction wether by simply giving it fuck all supplemented phosphate or by metabolic poisoning using arsenic has a large effect, indeed perhaps even a critical one, as alkaloid production is correlated with low levels of phosphate which retards growth and directs for alkaloid yielding.

Addition of a perfluorocarbon emulsion to the medium gives an increase in yield via increased uptake of O2, as does addition of more perfluorocarbon emulsion to the alginate solution the microbeads are made by.

Best way I see so far to make the beads is to use a syringe driver to spray a mixture of fungus, wether finely minced mycelium or spores, spores I think would be preferable as the mechanical stress involved in blending mycelium won't be taken lightly by the fungus and will really piss it off, through a fine needle, the finer the needle the smaller the beads, that has a 14KV electrical potential applied to it, I can't remember giving the needle positive and CaCl hardening solution negative or the reverse works best, but the electrostatic potential causes it to spray out as an ultrafine mist, which produces beads which harden on contact with the stirred calcium chloride solution.

I as yet find no article on doing so, but I plan to supplement the air intake with pure O2 from a cylinder, as factors concerning oxygen uptake matter greatly, increasing stirring and airation rate increase yields to a point, but with corresponding increased mechanical stress to the beads and their occupants, so by increasing the proportion of oxygen in the supplied air, one should be able to use less stirring and increase yields.

Addition of tweens to the medium of C.paspali cultures increases yield of paspalic acid via a surface tension effect allowing more nutrients to reach the fungus and be uptaken, radiolabelling studies show that the tween itself is not utilized in any way but merely alters surface tension so this effect should not, I believe, prove species specific./

Tryptophan acts as both an inducer of dimethylallyltryptophan synthase (DMAT) in the fungus which is one of hte first precursors of the ergoline ring system and also acts as a direct precursor of DMAT itself so addition of generous quantities of tryptophan will give it a further boost.

First step is a condensation of tryptophan, isopentenyl pyrophosphate (synthesis of the latter? ) and a methyl group donated from methionine, so I bet methionine addition will boost alkaloid synthesis some, as I believe, will mevalonic acid, a direct precursor to agroclavine.

TRYPTAMINE, 5-HYDROXY TRYPTOPHAN, 4-HYDROXY TRYPTOPHAN N-alpha-METHYLTRYPTOPHAN
N-alpha-METHYLTRYPTAMINE are not incorporated into agroclavine however according to this:

http://etd.lib.ttu.edu/theses/available/etd-12212009-31295015067209/unrestricted/31295015067209.pdf

Here is a thread that has a lot of useful material on it when it comes to genetic modifications specifically of plants, but the biotek.zip is good stuff IMO - but It sounds like embezzler would be better at making that call than I would be, and i am friends with the person who helped make it IIRC...
http://127.0.0.1/talk/index.php/topic,990.0.html

Ok, so let me know if this is the right track and what it is you are thinking...
it would go something like this:

First isolate the gene.
 this is done by:
perform DNA extraction of the ergot.
chop the DNA up with restriction enzymes.
perform electrophoresis to separate out the segments that are the correct length the gene should hopefully be...
determine if it is the correct gene -- how do you determine this?!
Make copies of the gene.
add the same restriction enzymes to the cell, cutting up some of its DNA, which will allow the gene to be inserted/attached into the fungi's DNA again. OR you can insert a plasmid of the gene into the cell?
 -- how is it one inserts genes into the living cells?  I know plants use agrobacterium, but fungi is different than plants...

I'll look more into it. keywords or a scribbled step by step like above (but better) are welcome...
Am I even on the right track? Seems pretty hard. Especially if the restriction enzymes have to be isolated as well from organisms -- though, that ought not be too hard? Probably easier to buy those..

Why not go with something to start with thats just on the high end of standard, like a 34g needle? paper I read less than that (30, 29 etc) gives a huge majority of beads under 100 microns in diameter, I think 50 micron beads would be perfect, 100 acceptable most likely, especially when co-hardened with perfluorocarbon aqueous emulsion instead of plain distilled water.

Just went out the other day and recovered these lil gems to start working on

Not sure how long really, but not THAT long, I had made an early appointment with my doc, and my housemate was still asleep, I left her a note, and went and got my scripts, decided to buy a drink, munch down a handfull of dihydrocodeine 30s and go picking ergot, and filling my face with wild blackberries.

How long, heh, couldn't tell you, I got lost in the autie hyperfocus, could have been half an hour, but in more likliehood it was about three and a half:D


That isn't half of what I have either, from other expeditions the other year I have a couple of medical piss-sample jars full of sclerotia from last year, another jar buried in a safe place, and some unusual, very thin, almost triangular, deeply grooved sclerotia of the same color as C.purpurea, but the gross differentiation in morphology suggests to me they may be another species, possibly C.sulcata.

Ageed, extant studies mainly concern C.purpurea, C.paspali, and there is a significant body of research on C.fusiformis which produces large quantity of the lower clavines but no lysergic acid derivatives whatsoever due to defective, yet still present, isoforms of the latter genes of the biosynthetic pathway.

Interestingly C.fusiformis strains appear to be far more stable as producers of their respective alkaloids than is C.purpurea.


I am wondering....chimaeras, people? hybridize high yielding (of both species, more precursors equals more lysergic acid via protoplast fusion, or parasexual reproduction, although that would present the problem of distinguishing between segregated parent strains and the resulting diploid fusion product.


One thing I do want to try , later in the progress of my magnum opus is to try and obtain at least some of the genes in the lysergic acid pathway and overexpress them in an already high yielding strain of C.purpurea, weather centrally via electroporesis, CaPO3 precipitation, viral transfection etc, or extra-nuclear-ly in plasmids, or hell, both to create the arnold schwartzenegger of the ergot world

cool hobby yes but from a practical point of view infected rye feilds are still the method of ergot production.

http://books.google.com/books?id=ef0tP77SdPYC&pg=PA345&lpg=PA345&dq=ergocristine&source=bl&ots=4lgqB4Pjm9&sig=8LE8OpSO8vp8IMLvNLp0mvFUIoU&hl=en&ei=EClbTP_lKIKB8gbjmozUAg&sa=X&oi=book_result&ct=result&resnum=10&ved=0CDEQ6AEwCTgU#v=onepage&q=ergocristine&f=false

hmm the book i referenced above outlines both methods of production. parasitic and saprophytic.
it's a nice read.

or say fuck all that and ring up one of those czeck suppliers

hmmm we might have a personal chat about that there are a few firms out there but by and large you are right most will sell you the stuff then drop a dime to customs.
but there are exceptions it all begins with a working relationship with the suppiler you can't just out and out say i'd like 100 grams of ergocristine please.
you have to buid a business realtionship with them to first see if they misdeclare for tax purposes and shit like that.
you know while i'm on the topic one of you research monkeys could retrieve an article from the royal society of chemistry concerning adding diethylamine to the fermenting broth to produce lsd directly from the saprophytic culture.
i looked and looked but i suspect the feds pulled it.