Limpet_Chicken
Bluelighter
- Joined
- Oct 13, 2005
- Messages
- 6,323
Oh and taco, I just noticed your post about ergot. Actually I am gearing up to work with it. Although I DEFINITELY do NOT intend on consuming any extract of it directly. Rather, plan is to isolate a production strain, and keep on with the mutagenesis until one that produces stably and reliably upon subcultures (even better would be a conidia-producing,alkaloid productive strain, which is quite rare although not impossible.
Probably means I'm going to have to go through an awful bloody lot of petri dishes full of agar before I get anywhere, although even alkaloid high producers that aren't stable in subculture, I'll still take the alkaloids I can get from such strains before they poop out.
And I've been doing a LOT of research and reading in the scientific literature already out there, as well as a few experiments of my own, such as starting with a conidiating strain thats stable but not much of an alkaloid producer, and both protoplast-fusion with cell lines from the same strain, since it seems heterokaryotic strains are more likely to be alkaloid producers than monokaryotic lines, as well as treatment with colchicine and with mitogenic agents such as pokeweed toxin, already got my sclerotiae just need to get the likes of a lot of mannitol (they seem to thrive particularly on mannitol, and unfortunately glucose can't be used, at least in Claviceps purpurea, as the fungus turns it into polysaccharide glorp that buggers up oxygen transport. Which fouls up the growth medium and causes a big loss in alkaloid production. Even more of a problem since I intend on using alginate polymer microspheres, produced via electrostatic spraying into a CaCl2 solution [they can not only survive extremely high osmotic pressures but thrive in them, when the osmotic pressure is from sugars as nutrients, its quite extreme, their tolerance for it, so much so that at least one researcher in the history of Claviceps research, according to Kren and Cvak et. al. in the book 'The Genus Claviceps', thought that '300g sucrose per liter' was a typo, and that 30g was meant. It wasn't. They meant 300g.)
And of course, after experiments with shake-flask culture and various innoculation vs production medium (they need high phosphate media for good, vigorous growth but alkaloid production is dependent upon low phosphate levels. Arsenical compounds can be applied carefully, in small quantities as a metabolic poison to increase alkaloid yield, as it interferes with phosphate utilization being chemically similar to phosphorus, ideally, as arsenate. And IIRC to save money, 10% of the mannitol can be substituted for potassium chloride to maintain the osmotic balance, and save costs, being cheaper than mannitol, and it seems like asparagine is an ideal nitrogen source. Encapsulation in alginate microspheres serves both as a mechanical buffer, to prevent wear and tear type damage, because Claviceps spp. are rather sensitive to mechanical stress, and also to simulate an artificial pseudosclerotia type condition. The microspheres need to be as small as possible, because anything more than a couple of millimeters there becomes an anoxic internal zone where whilst the fungus located there might survive, the oxygen required to produce alkaloids higher than clavines is unavailable. So use of an oxygen-carrying perfluorocarbon emulsion is planned, as is addition of tween, as a surfectant which reportedly improves cellular permeability to O2. Might try extraction of the haemoglobin from a pint or so of my blood, and addition of the haemoglobin along with the perfluorocarbon emulsion base for the polyalginate microspheres, and contrast it with perfluorocarbon emulsion alone, haemoglobin alone and both.
The perfluorocarbon emulsion base for microsphere encapsulation does work, that much is known. I don't know if any experiments including haemoglobin have ever been performed by others though. I've got plenty ideas to try, along with of course the well known tricks and enhancements for improving alkaloid productivity.
Probably means I'm going to have to go through an awful bloody lot of petri dishes full of agar before I get anywhere, although even alkaloid high producers that aren't stable in subculture, I'll still take the alkaloids I can get from such strains before they poop out.
And I've been doing a LOT of research and reading in the scientific literature already out there, as well as a few experiments of my own, such as starting with a conidiating strain thats stable but not much of an alkaloid producer, and both protoplast-fusion with cell lines from the same strain, since it seems heterokaryotic strains are more likely to be alkaloid producers than monokaryotic lines, as well as treatment with colchicine and with mitogenic agents such as pokeweed toxin, already got my sclerotiae just need to get the likes of a lot of mannitol (they seem to thrive particularly on mannitol, and unfortunately glucose can't be used, at least in Claviceps purpurea, as the fungus turns it into polysaccharide glorp that buggers up oxygen transport. Which fouls up the growth medium and causes a big loss in alkaloid production. Even more of a problem since I intend on using alginate polymer microspheres, produced via electrostatic spraying into a CaCl2 solution [they can not only survive extremely high osmotic pressures but thrive in them, when the osmotic pressure is from sugars as nutrients, its quite extreme, their tolerance for it, so much so that at least one researcher in the history of Claviceps research, according to Kren and Cvak et. al. in the book 'The Genus Claviceps', thought that '300g sucrose per liter' was a typo, and that 30g was meant. It wasn't. They meant 300g.)
And of course, after experiments with shake-flask culture and various innoculation vs production medium (they need high phosphate media for good, vigorous growth but alkaloid production is dependent upon low phosphate levels. Arsenical compounds can be applied carefully, in small quantities as a metabolic poison to increase alkaloid yield, as it interferes with phosphate utilization being chemically similar to phosphorus, ideally, as arsenate. And IIRC to save money, 10% of the mannitol can be substituted for potassium chloride to maintain the osmotic balance, and save costs, being cheaper than mannitol, and it seems like asparagine is an ideal nitrogen source. Encapsulation in alginate microspheres serves both as a mechanical buffer, to prevent wear and tear type damage, because Claviceps spp. are rather sensitive to mechanical stress, and also to simulate an artificial pseudosclerotia type condition. The microspheres need to be as small as possible, because anything more than a couple of millimeters there becomes an anoxic internal zone where whilst the fungus located there might survive, the oxygen required to produce alkaloids higher than clavines is unavailable. So use of an oxygen-carrying perfluorocarbon emulsion is planned, as is addition of tween, as a surfectant which reportedly improves cellular permeability to O2. Might try extraction of the haemoglobin from a pint or so of my blood, and addition of the haemoglobin along with the perfluorocarbon emulsion base for the polyalginate microspheres, and contrast it with perfluorocarbon emulsion alone, haemoglobin alone and both.
The perfluorocarbon emulsion base for microsphere encapsulation does work, that much is known. I don't know if any experiments including haemoglobin have ever been performed by others though. I've got plenty ideas to try, along with of course the well known tricks and enhancements for improving alkaloid productivity.