ampicillin is worth a shot
Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #80 on: April 06, 2011, 05:03:08 PM »
Topic: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali) (Read 1616 times)
aniracetam
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Tsathoggua
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #81 on: April 06, 2011, 06:14:31 PM »
Much rather not, personally, if I can help it. Chloramphenicol will most likely be my antibiotic of choice.
I do have rather a lot of various different penicillin variants hanging about that I saved for use keeping plates free of contamination, that were scripted, but not taken, by the goat-raping Bitch Queen woman when I gave her fat arse the heave ho out of here, but there is the nasty snag of my being allergic, I would have to purify the contents of the capsules etc, and suffice as to say, whilst normally fairly benign stuff, I personally would have to handle the operation with extreme care, gas mask, arm length gloves job for me.
Snagged a fair few asthma rescue inhalers for that reason, I won't work with the penicillins unless I can't find anything else, and then predose with antihistamines, and keep an inhaler by my side in case of emergency, ironically, the antibiotics are probably more toxic than the ergot in my case :p worth a shot? maybe, but there is the possibility, if I fucked up, of it being a shot in the arse from an epi-pen.
I do have rather a lot of various different penicillin variants hanging about that I saved for use keeping plates free of contamination, that were scripted, but not taken, by the goat-raping Bitch Queen woman when I gave her fat arse the heave ho out of here, but there is the nasty snag of my being allergic, I would have to purify the contents of the capsules etc, and suffice as to say, whilst normally fairly benign stuff, I personally would have to handle the operation with extreme care, gas mask, arm length gloves job for me.
Snagged a fair few asthma rescue inhalers for that reason, I won't work with the penicillins unless I can't find anything else, and then predose with antihistamines, and keep an inhaler by my side in case of emergency, ironically, the antibiotics are probably more toxic than the ergot in my case :p worth a shot? maybe, but there is the possibility, if I fucked up, of it being a shot in the arse from an epi-pen.
aniracetam
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #82 on: April 06, 2011, 07:24:25 PM »
ehh, i'm going with 4 mL amp/L culture, 5 mg/mL conc.
seed culture is already in its prime
seed culture is already in its prime
Vesp
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #83 on: April 06, 2011, 08:22:06 PM »
I wish you the best of luck with everything! I'm sure the info you gather will be gold!
Bluebottle
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #84 on: April 10, 2011, 04:37:42 PM »
While Gilmanov apparently found hydrocarbons helpful (I don't have the article), Application of oxygen vectors to Claviceps purpurea cultivation (Menge et al, 2000) found useless, even toxic. Instead, perfluorocarbons were shown to have potential, but even so, "Although the shakeflask results were promising, the bioreactor studies showed no positive effect of adding PFC to the fermentation medium."
Admittedly, an emulsifier would have helped, and they didn't use one. But, even olive oil dissolves oxygen about 3x better than water does, vs. 8x for toluene, and it's probably less toxic. (Liquid paraffin actually works less well than olive oil, not sure about other natural oils, silicone might work) It might be a hassle to separate the emulsion, but I'm sure it could be done if any benefit were found.
The other thing that occurs to me is (if an emulsifier isn't used) the hydrophobic phase could be regularly removed, extracted with acidic water, and recycled. I'm pretty sure the ergopeptides are fairly lipophilic, and decreasing their concentration in the stock might just get them to make even more. But, just an idea.
It would be fantastic if EGCG or curcumin worked, I still think garcinol itself would be worth finding/trying. If it pans out, not least would be the ability to use pretty much any wild strain, as well as other points already mentioned. Hell, the DEA'd throw a fit if you could strip down the medium to tap water, karo syrup, turmeric and lemon juice, haha, well a man can dream.
Admittedly, an emulsifier would have helped, and they didn't use one. But, even olive oil dissolves oxygen about 3x better than water does, vs. 8x for toluene, and it's probably less toxic. (Liquid paraffin actually works less well than olive oil, not sure about other natural oils, silicone might work) It might be a hassle to separate the emulsion, but I'm sure it could be done if any benefit were found.
The other thing that occurs to me is (if an emulsifier isn't used) the hydrophobic phase could be regularly removed, extracted with acidic water, and recycled. I'm pretty sure the ergopeptides are fairly lipophilic, and decreasing their concentration in the stock might just get them to make even more. But, just an idea.
It would be fantastic if EGCG or curcumin worked, I still think garcinol itself would be worth finding/trying. If it pans out, not least would be the ability to use pretty much any wild strain, as well as other points already mentioned. Hell, the DEA'd throw a fit if you could strip down the medium to tap water, karo syrup, turmeric and lemon juice, haha, well a man can dream.
overunity33
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #85 on: April 10, 2011, 06:14:57 PM »
While Gilmanov apparently found hydrocarbons helpful (I don't have the article), Application of oxygen vectors to Claviceps purpurea cultivation (Menge et al, 2000) found useless, even toxic. Instead, perfluorocarbons were shown to have potential, but even so, "Although the shakeflask results were promising, the bioreactor studies showed no positive effect of adding PFC to the fermentation medium."
Admittedly, an emulsifier would have helped, and they didn't use one. But, even olive oil dissolves oxygen about 3x better than water does, vs. 8x for toluene, and it's probably less toxic. (Liquid paraffin actually works less well than olive oil, not sure about other natural oils, silicone might work) It might be a hassle to separate the emulsion, but I'm sure it could be done if any benefit were found.
The other thing that occurs to me is (if an emulsifier isn't used) the hydrophobic phase could be regularly removed, extracted with acidic water, and recycled. I'm pretty sure the ergopeptides are fairly lipophilic, and decreasing their concentration in the stock might just get them to make even more. But, just an idea.
It would be fantastic if EGCG or curcumin worked, I still think garcinol itself would be worth finding/trying. If it pans out, not least would be the ability to use pretty much any wild strain, as well as other points already mentioned. Hell, the DEA'd throw a fit if you could strip down the medium to tap water, karo syrup, turmeric and lemon juice, haha, well a man can dream.
PFC's are expensive as hell, although available to the average consumer... The problem is the huge volume required (i think like 20% volume +)
interesting about a 2 phase system, it would have to be tried experimentally because it will also remove lipids etc, all these things are involved in signaling for alkaloid production.
EGCG/curcumin is top priority, if you can skip straight to gene expression then there is no reason to have the culture medium that we do or bother with high yielding strains... I know that the high osmotic pressure in these culture mediums signals that the fungus has broken through the plant into its nutrient system, so thats one thing that will not be necessary if these work.
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #86 on: April 10, 2011, 08:39:33 PM »
Immobilization of PFC within alginate microspheres is possibly the best way to go, probably don't need as much PFC that way, and its highly localized to the Claviceps cells, sealed there within the matrix.
I'm guessing high osmotic pressure in the medium will be advantageous anyhow, regardless of other fractors, not that many other nasty competitive organisms are going to thrive too well in there in a highly concentrated sugar/electrolyte soup.
I'm guessing high osmotic pressure in the medium will be advantageous anyhow, regardless of other fractors, not that many other nasty competitive organisms are going to thrive too well in there in a highly concentrated sugar/electrolyte soup.
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #87 on: April 11, 2011, 03:04:02 AM »
Quote
It would be fantastic if EGCG or curcumin worked, I still think garcinol itself would be worth finding/trying. If it pans out, not least would be the ability to use pretty much any wild strain, as well as other points already mentioned. Hell, the DEA'd throw a fit if you could strip down the medium to tap water, karo syrup, turmeric and lemon juice, haha, well a man can dream.
Quote
EGCG/curcumin is top priority, if you can skip straight to gene expression then there is no reason to have the culture medium that we do or bother with high yielding strains... I know that the high osmotic pressure in these culture mediums signals that the fungus has broken through the plant into its nutrient system, so thats one thing that will not be necessary if these work.
Check out these structures that makes the EGCG/Curcumin Hypothesis seem much legit
Garcinol - A HATi (histone Acetylase Inhibitor) -- Confirmed to induce/increase Ergot Alkaloid Synthesis in Claviceps, might work with other members of the same family.
EGCG
Curcumin
Histone Acetyltransferase Inhibitor II
2,6-bis-(3-Bromo-4-hydroxybenzylidene)cyclohexanone
EGCG
Curcumin
Histone Acetyltransferase Inhibitor II
2,6-bis-(3-Bromo-4-hydroxybenzylidene)cyclohexanone
I think the structures of these three show a very similar backbone - a hydrophilic oxy group on the left hand side - either top or bottom? probably matters.. but the molecules can bend a bit, right?
An oxy/hydrophilic portion in the center and on the top.
and... than it starts to get confusing and the molecules vary widely....
But these images and the sorta reoccurring structural patterns make this look good!
Of course, also others that are NOT active, or do the OPPOSITE effect of what we want also look similiar - however th curcumin and ECGC are known HATis - so from what i can tell, having absolutely no education in any of this, it looks pretty good.
aniracetam
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #88 on: April 11, 2011, 05:37:49 PM »
so what sort of ratios of EGCG, circumin, Karo, and citric acid are we talking here?
i propose:
5% Karo
2% EGCG
1% Circumin
2% Citric acid
i propose:
5% Karo
2% EGCG
1% Circumin
2% Citric acid
Vesp
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #89 on: April 11, 2011, 10:35:36 PM »
I say test the EGCG alone in karo water, and curcumin in karo water -- than possibly though I think less meaningful is ECGC+Curcumin in karo water...
Hopefully the ECGC or the curcumin is active, or both active... and not need any combination of one another...
and again ECGC is heat sensitive so be careful about getting it to hot - a brief boil ought to do the trick. Esp. if the concentration of sugar is very high to inhibit the growth of any possible contaminations...
Hopefully the ECGC or the curcumin is active, or both active... and not need any combination of one another...
and again ECGC is heat sensitive so be careful about getting it to hot - a brief boil ought to do the trick. Esp. if the concentration of sugar is very high to inhibit the growth of any possible contaminations...
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #90 on: April 12, 2011, 05:05:40 PM »
So its pretty easy to figure out if your system is limited by the dissolved oxygen, which is important in optimizing and simplifying your setup. If your DO is just fine and you have a stirrer, take it out, maybe you can get away without it, or if you have a weak air system you may be able to get away with increased stirring as long as the DO tests OK. Its interesting how you can research information on submerged culturing of c. paspali to quite an extent, but also c. purpurea literature often applys, then you can move on the c. fusiforms (sp?) then antibiotic producing reactors, it really works similarly across the board.
" The rotary shakers employed for all submerged flask cultures operated at 200 rev./min with a 9 cm eccentric throw. Under these conditions, an oxygen transfer rate of 38 ml oxygen (at s.t.p.)/Ioo ml/h was recorded by means of the sulphite oxidation technique of Cooper, Fernstrom & Miller (I 944)."
"The fact that the dissolved oxygen concentration never fell below35%of the saturation value suggeststhat the oxygen supply was not limiting at any stage."
COOPERC,.M.,FERNSTROGM.A,.&MILLERS,.A.(1944).Gas-liquidcontactors.IndustrialEngineering Chemistry 36, 504-509.
from
http://mic.sgmjournals.org/cgi/reprint/82/2/345.pdf
" The rotary shakers employed for all submerged flask cultures operated at 200 rev./min with a 9 cm eccentric throw. Under these conditions, an oxygen transfer rate of 38 ml oxygen (at s.t.p.)/Ioo ml/h was recorded by means of the sulphite oxidation technique of Cooper, Fernstrom & Miller (I 944)."
"The fact that the dissolved oxygen concentration never fell below35%of the saturation value suggeststhat the oxygen supply was not limiting at any stage."
COOPERC,.M.,FERNSTROGM.A,.&MILLERS,.A.(1944).Gas-liquidcontactors.IndustrialEngineering Chemistry 36, 504-509.
from
http://mic.sgmjournals.org/cgi/reprint/82/2/345.pdf
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #91 on: April 12, 2011, 07:32:04 PM »
C.fusiformis doesn't produce the ergopeptides we are after...
The gene cluster for EA synthesis is present, but at least one gene (DMAW I think but I can't remember off the top of my head) is present in a non-functional isoform and won't produce past one of the later stage clavines.
The gene cluster for EA synthesis is present, but at least one gene (DMAW I think but I can't remember off the top of my head) is present in a non-functional isoform and won't produce past one of the later stage clavines.
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #92 on: April 12, 2011, 07:48:41 PM »
C.fusiformis doesn't produce the ergopeptides we are after...
The gene cluster for EA synthesis is present, but at least one gene (DMAW I think but I can't remember off the top of my head) is present in a non-functional isoform and won't produce past one of the later stage clavines.
Im aware of all that, Im just saying similar techniques and considerations are used... especially on the subject of system design
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #93 on: April 12, 2011, 07:53:29 PM »
is there anything in the lit about supplying too much O2?
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #94 on: April 12, 2011, 07:59:10 PM »
Keep in mind that turmeric has about 0.58 to 3% of curcumin itself, and presumably various nutrients.
On the note of chemical sterilisation, H2O2 and catalase might be worth a try. I imagine mashing a potato in peroxide solution would sterilise itself, and make a suitable solution of the enzyme.
Too much O2 seems like a possibility hypothetically, but if it were easy to reach I guess there wouldn't be so many attempts to introduce as much of it as possible.
O2 has a fairly low solubility in water. The alkaloids do degrade over time for whatever reason, and oxidation probably does its part in that, so there is definitely a proper time to harvest.
On the note of chemical sterilisation, H2O2 and catalase might be worth a try. I imagine mashing a potato in peroxide solution would sterilise itself, and make a suitable solution of the enzyme.
Too much O2 seems like a possibility hypothetically, but if it were easy to reach I guess there wouldn't be so many attempts to introduce as much of it as possible.
O2 has a fairly low solubility in water. The alkaloids do degrade over time for whatever reason, and oxidation probably does its part in that, so there is definitely a proper time to harvest.
aniracetam
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #95 on: April 12, 2011, 08:17:53 PM »
i don't see oxidation being much of an issue in solution saturated with antioxidative acids such as citric or succinic,
at least not in the short term. of course, using amber glass also helps
edit: amp. is effective as the antibiotic
at least not in the short term. of course, using amber glass also helps
edit: amp. is effective as the antibiotic
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #96 on: April 13, 2011, 03:37:33 AM »
to much o2 really isnt a problem, there is a optimal time to harvest, the alkaloids slowly assume other forms which I suspect would still yield lysergic acid on hydrolysis, someone correct me if im wrong.
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #97 on: April 13, 2011, 03:42:15 AM »
you're correct
for the stevens and hall strain, it's mostly going to be paspalic acid, then lsa
for the stevens and hall strain, it's mostly going to be paspalic acid, then lsa
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #98 on: April 13, 2011, 05:20:30 PM »
Quote
Polyisoprenylated Benzophenone, Garcinol, a Natural Histone Acetyltransferase Inhibitor, Represses Chromatin Transcription and Alters Global Gene Expression* DOI 10.1074/jbc.M402839200
"In agreement with the results of the p300 filter binding assay, it was found that the HAT activity of PCAF was almost completely inhibited by 10 uM garcinol compared with the Me2SO control, whereas even at 20 uM concentration, 5–10% of 3H-labeled histone H3 could be detected. Interestingly it was found that acetylation of histone H4 by p300 was more sensitive to inhibition by garcinol compared with that of H3. The p300-mediated acetylation of histone H4 was completely inhibited at a 1 uM concentration of garcinol, while 20 uM could not abolish the acetylation of H3."
"Garcinol-mediated inhibition kinetics (for both p300 and PCAF) shows that with changing concentrations of acetyl-CoA it behaves like an uncompetitive type of inhibitor whereas for core histones, as a competitive inhibitor.... garcinol specifically inhibits HAT activity-dependent chromatin transcription but not transcription from the DNA template."
"There are very few HAT inhibitors known to date. The first reported HAT inhibitors were bisubstrate (29) types of inhibitors of p300 and PCAF, which contain CoA moieties. One of these compounds, Lys-CoA has proven useful for blocking the HAT activity of p300 specifically. Though it has been employed ...with the use of cellpermeabilizing agents (41), Lys-CoA has generally been ineffective with simple addition to cell culture media."
"Recently we have isolated the first naturally occurring HAT inhibitor, anacardic acid (AA), which inhibits the HAT activity of both p300 and PCAF very effectively (31). By using AA as a synthon we have synthesized an amide derivative of anacardic acid CTPB, which is the only known small molecule activator of any histone acetyltransferase (p300). Significantly, CTPB is exclusively specific for p300 HAT activity. However cells are not permeable or poorly permeable to both anacardic acid and CTPB."
http://en.wikipedia.org/wiki/Anacardic_acid
http://www.ncbi.nlm.nih.gov/pubmed/18487348
Incidentally, "Stoppage of fungal growth in the rachilla of C. purpurea has been shown to be most likely caused by host phenolics that accumulate during infection at this site." (Biology and Life Strategy of the Ergot Fungi Klaus B. Tenberge) So ever since I read that I had been curious what effect phenolic substances, tannins perhaps, have on the alkaloid production of a claviceps culture. And it strikes me that the HATi mentioned (Garcinol, EGCG, Curcumin, Anacardic acid) are all prominently phenolic! Perhaps a coincidence, but I wonder what a dose of the humble salicylic acid would do?
Polyisoprenylated Benzophenone Garcinol a Natural HATi.pdf
(691.5 kB - downloaded 4 times.)Vesp
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Re: Ergot Culture Medium for Alkaloid Production (C. Purpurea & Paspali)
« Reply #99 on: April 13, 2011, 07:54:15 PM »
Wow! Great observations Bluebottle!!!
It seems like what might be better to test is various Urushiols - http://en.wikipedia.org/wiki/Urushiol
That is a phenolic compound and has a very similiar structure to many of the HATis
Perhaps to get a "natural amount" of ergot alkaloids one might want to look into
So yeah - perhaps there it is! Cheap, Unregulatable, OTC source of whatever compounds make them produce alkaloids - Just need to find a way to extract it from rye, wheat, or barely.
It might be good, if at all possible to find the percent of ergot alkaloids in the sclerotia relative to their host plant -- that might indicate the best source for these compounds. But maybe not.
It seems like what might be better to test is various Urushiols - http://en.wikipedia.org/wiki/Urushiol
That is a phenolic compound and has a very similiar structure to many of the HATis
Perhaps to get a "natural amount" of ergot alkaloids one might want to look into
Quote
In the major cereals, alkylresorcinols have been reported to be present in high levels (> 500 mg/g) in wheat and rye and in lower amounts in barley and millet. They are found mainly in the outer layers (bran fraction) of cereal grains. The homologue composition also varies greatly across the species; barley has the highest proportion of C25:0 (about 40%), rye has the highest proportion of C19:0 (about 30%), and wheat has the highest proportion of C21:0 (about 50%). Alkenylresorcinols are also present in cereals. Their amounts vary between 0.5% for barley, 1% for wheat, and 15% for rye. Not only the amounts but also the compositions of the specific mixtures are different. Thus, the ratio of the homologues C17:0/C21:0 is about 0.1 in wheat and 1.0 in rye, indicating that this determination may be used as good markers of these cereal products (Chen Y et al., J Agric Food Chem 2004, 52, 8242).--- http://www.cyberlipid.org/simple/simp0006.htm
So yeah - perhaps there it is! Cheap, Unregulatable, OTC source of whatever compounds make them produce alkaloids - Just need to find a way to extract it from rye, wheat, or barely.
It might be good, if at all possible to find the percent of ergot alkaloids in the sclerotia relative to their host plant -- that might indicate the best source for these compounds. But maybe not.