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Author Topic: Adding haemoglobin to alginate imobilised cells to boost O2 transport?  (Read 32 times)

Tsathoggua

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Adding haemoglobin to alginate imobilised cells to boost O2 transport?
« on: May 11, 2010, 05:21:36 PM »
What do the good bees'n'girls here think of the idea of adding haemoglobin to alginate microparticles containing immobilised cells?

Claviceps being the organism in question here, in submerged culture.
Of course, harmoglobin could be isolated in gram quantities I would have thought quite easily, its not as if one cannot spare a pint or two of ye olde human-juice, and much much more could be obtained perhaps if one is up to organising a little donation drive and offering a few quid to a few friends per pint :D

So, how about it? co-immobilizing the Claviceps cells in the alginate beads with haemoglobin to improve oxygen uptake and allow the beads to synthesize things more useful than clavines deeper into the microparticles, with or without fluorocarbon oxygen vector emulsions also coimmobilized thusly?
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Vesp

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Re: Adding haemoglobin to alginate imobilised cells to boost O2 transport?
« Reply #1 on: May 12, 2010, 12:32:41 AM »
Crazy idea, seems like it would cause problems with the ergot.
 
Instead of using your own blood, you could just get blood from a butcher shop.
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embezzler

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Re: Adding haemoglobin to alginate imobilised cells to boost O2 transport?
« Reply #2 on: May 12, 2010, 10:05:18 AM »
The erythrocytes will degrade too quickly, they do not have a very long life in vivo anyway but in the presence of the claviceps I would expect the cells and especially the haemaglobin chains to be broken pretty quickly. Also the physiological pH which allows for haem to bind oxygen may not be the same as your culture pH so it may not work at all.

I would be very reluctant to use even well centrifuged and seperated blood since it will still contain leukocytes and secreted immune proteins like antibodies and complement proteins. It is nearly the last thing I would want in the mix truth be told. Often sheeps blood agar is used for solid culture of organisms but this is to provide nutrients not to enhance oxygen uptake as it would have been autoclaved.

I would look at keeping the alginate bead size lower (giving greater surface area and exposure to the liquid media) to enhance oxygen transfer since you are looking for an immobilised culture but my main focus would be on getting something growing and worry about yield afterwards.

Appropriate agitation of the liquid phase would be your best bet for keeping the oxygen levels high enough but I would not be using blood for this experiment.

Have you found a correct culture media for the claviceps? The most important thing would be to get something growing and then worry about yield.

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Tsathoggua

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Re: Adding haemoglobin to alginate imobilised cells to boost O2 transport?
« Reply #3 on: May 12, 2010, 01:39:28 PM »
Low phosphate is seemingly one of the most important things as far as yield is concerned, and there are many, many examples of culture mediums around, I'm still looking at some studies on trace element nutrition as far as restriction and supplementation goes.

And I didn't mean red blood cells, but purified haemoglobin protein itself, I want to learn some techniques for working with DNA and the like, and that will include electrophoresis, seperating a few proteins out would give me the ideal chance to test out the electrophoresis chamber I am building.

Keeping size down is essential, especially after the growth phase is done with, and the beads are clogged with hyphae, oxygen transport goes straight down the shitter, I'll probably be using a perfluorocarbon emulsion of some variety to enhance O2 uptake, and perhaps a slightly enriched air supply, increasing stirring rate/air pump output does work to a degree, but Claviceps is quite sensitive to being disturbed, lil guys don't like being treated roughly, it drops yield of lysergic acid and the lysergyl peptides considerably.

Quite possibly butchers won't sell blood here, I seem to recall problems getting it when I kept pet leeches years back, nearly everywhere refused to sell/give away blood citing elf'n'safety BS.

One idea I had, to do the actual forming of the beads was to use one of those pump-misters intended for horticulture that put out a really fine mist to spray the alginate/perfluorocarbon/whatever else goes in to the beads into the CaCl2 hardening solution.
I read a couple of papers on alginate immobilisation where they use a small ioniser to further optimise, resulting in enhanced production in Claviceps cells.
« Last Edit: May 12, 2010, 01:59:48 PM by Tsathoggua »
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poisoninthestain

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Re: Adding haemoglobin to alginate imobilised cells to boost O2 transport?
« Reply #4 on: May 12, 2010, 07:02:05 PM »
Hemoglobin is going to hemolyze on exposure to O2 and CO2 into heme and globin in under 30 minutes if you're using blood samples from pig or man. The globin part is further going to break down into a lipid-soluble protein called unconjugated bilirubin, then around ~45min after UV light exposure it will lose it's lipid affinity and become water soluble as photobilirubin. Ever see jaundice? That's bilirubin. The ergot's gonna turn yellow and the hemoglobin for added O2 is gonna be useless. Blood PH is 7.35-7.45.  :P Nurse PStain.
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