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Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination
    #7914810 -

Hey all,

I made a new account here for security reasons. I work in a lab that specializes in bioluminescence, mostly focusing on firefly luciferase and related beetles. Recently we have decided to branch out a bit into other organisms for some ideas for 2008 research projects. One of these ideas concerns the isolation and structural determination of unknown luciferin(s) in bioluminescent fungi.

After doing some internet research I found there was a general consent that Panellus stipticus was not only easy to grow, but the brightest of the luminous species. I searched through past threads here on shroomery concerning the cultivation of this mushroom which lead me to some grow-kits on sporeworks. These kits look great and my lab is ready to purchase several of them.

I was wondering if there is anyone out there who has had success in growing Panellus, and if so, if there were any special conditions that were required for colonization, fruiting, etc that would help us maximize the success of our research. From what I've read so far, growing this mushroom seems very straightforward and basic. The mycelium is said to glow much less than the fruit bodies. This means the kits would have to be grown all the way through fruiting for the best chances at gaining some knowledge out of this project. I'm just looking for personal experiences that might help us out right off the bat, even if it's something along the lines of, "yeah i grew it, no problem, it glowed."

Wish us luck!

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: GlowMe]
    #7917567 -

GourmetMushroom did some work on Panellus stipticus not too long ago. I couldn't find the thread easily since they moved it into the growlog section.

I'm not sure if you can access that section yet. I think you might have to hit a certain length of membership or a certain post count before it becomes visible.

Here's a link...
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/6622398

Perhaps one of the mods could help with that.

It's interesting that you work with bioluminescence. There have been several requests for making various species glow over the years. If GE is something your lab also does that would certainly be an interesting project.

Any idea on the toxicity of various luciferases?


-FF

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: fastfred]
    #7917935 -

Pan stips are fun. Add a touch of lemon or orange peels to lower the pH, and they really glow like mad.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: RogerRabbit]
    #7919394 -

FF - Thank you so much for that link. Both the thread and the attached paper are extremely helpful. I accessed it with no problem using my normal account. It would indeed be interesting to be able to make other non-luminescent species of mushrooms glow. However there are a few problems (as of now) that would prevent this from happening. The first and most obvious is that both the luciferins and luciferases responsible for the production of light remain unidentified. Before the insertion of the gene into other species could occur, that gene itself would have to be found and cloned. The second major problem is the fact that there are many more proteins required for the reaction that just the luciferase. You would also need to find, sequence, and clone a whole range of enzymes responsible for producing the luciferins, their precursors, and any other co-ligands that might be required for the reaction. Then all these enzymes would have to be expressed at appropriate levels. A protein like GFP (green-fluorescent protein) can easily be cloned into cats, pigs, rabbits, mice, etc etc because it requires no substrate. It simply contains a chromophore that absorbs UV light and emits in the visible range. Firefly luciferase has been successfully cloned into plants and mice, but an injection of luciferin is required before any light is produced. As for the toxicity of the luciferases, I'm certain there would be none. You would digest it just like you would any other protein. Eating a few fireflies is not going to do you any harm other than making your teeth glow for a few minutes.

RR - As this is my lab's first foray into the growth of fungi I'm not too keen on presenting my knowledge of cultivation (ha) and thus this project will rely on the 3lb sawdust kits from sporeworks. If the project fails or continues too slowly, I may present my "findings" on making our own substrate. After reading gourmet's thread and the paper I realize that the substrate should have a pH of ~3.5-4, but supplementing sporeworks' already sealed and prepared bag will be impossible. The most I can hope for is decent colonization of the sawdust, followed by dunking and misting with pH 3.5 water. The pH optimum is extremely intriguing however. Not only for the fact that growth is most rapid at such a high acidity, but that the production of light itself is increased. This suggest a certain required protonation of the luciferin and is itself a clue to the compound's structure. The requirement of oxygen to the reaction is similar to that of firefly luciferase, which also needs O2 to produce light.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: GlowMe]
    #7919832 -

just talk to the guys over at sporeworks, then when your buddys at work ask where you got the idea, say sporeworks told you,

im not tryin to take credit away from RR, just offering him an idea as to how to explain without exposing himself


--------------------
We were sent to hell for eternity :hellfire: Ø:omgawesome:h®
We play on earth to pass the time :foreheadslap:

Over-population the root of all Evil-brings the Elites Closer to the gates.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination *DELETED* [Re: makaveli8x8]
    #7920308 -

Post deleted by buddhahoodlum

Reason for deletion: dee dee dee



--------------------
“I believe that water will one day be employed as fuel, that hydrogen and oxygen which constitute it, used singly or together, will furnish an inexhaustible source of heat and light, of an intensity of which coal is not capable.” – Jules Verne, T

“Ere many generations pass, our machinery will be driven by power obtainable at any point in the universe...it is a mere question of time when men will succeed in attaching their machinery to the very wheel-work of nature."
- Nikola Tesla


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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: buddhahoodlum]
    #7920810 -

buddhahoodlum said:
supposed to add veg oil,kelp,stevia,gypsum to your compost.


Really now? While kelp, stevia and a small amount of vegetable oil would do no harm, they're certainly not necessary or something you're 'supposed' to do. Be nice.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: buddhahoodlum]
    #7920865 -

did you read glowme's post??? please do again and then you'll realize its you being a tard. and the fact that you remember that seaweed and oil crap from god knows how long ago is more proof of it.

just to clarify, glowme has "security" issues, i suggested he say he talked to sporeworks.


--------------------
We were sent to hell for eternity :hellfire: Ø:omgawesome:h®
We play on earth to pass the time :foreheadslap:

Over-population the root of all Evil-brings the Elites Closer to the gates.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: buddhahoodlum]
    #7920880 -

buddhahoodlum said:
Makaveli You didn't even know that you were supposed to add veg oil,kelp,stevia,gypsum to your compost.  Try harder at not being tarded next time.


MORON!! :spank:

You sound like a 17year old kid that knows everything about growing mushrooms.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination *DELETED* [Re: derx]
    #7920974 -

Post deleted by buddhahoodlum

Reason for deletion: dee dee dee


Edited by buddhahoodlum (01/23/08 08:07 PM)

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: buddhahoodlum]
    #7921507 -

Yes, I have grown these. I also brought a big nice aquarium of them to Burning Man 2007, only for it to be blow to pieces by storms.... ;( It was totally amazing before I left, though. Stpiticus likes acidic conditions. Growing around 3.8 to 4.0 pH stimulates faster growth and also brighter glowing. I was using alder sawdust, oak chips, rice bran, and gypsum. The mix was pH-adjusted down to 3.8 using citric acid crystals (you can get them at any brewery supply). It fruited like mad indoors at room temperatures and close to 100% RH. You can do a search to read about it.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: buddhahoodlum]
    #7921871 -

Quote:
RR - As this is my lab's first foray into the growth of fungi I'm not too keen on presenting my knowledge of cultivation (ha) and thus this project will rely on the 3lb sawdust kits from sporeworks. If the project fails or continues too slowly, I may present my "findings" on making our own substrate. 
but supplementing sporeworks' already sealed and prepared bag will be impossible.



this is what I was responding to when I made my first post.

The suggestion i made to him was to just say he talked to sporeworks and thats how he came up with his findings for getting them brighter or w/e...which for "security reasons" is better than saying he was on the shroomery and already knows how to cultivate...which ive bolded all of this up above.

so no I'm not retarded and no I'm not paranoid either...well sometimes i am but this wasn't one of them :tongue:


back on topic, Gormetmushrooms what made you decide to use citric acid crystles and were they better than lemons?  I got a jar of stips goin right now as well, about to spawn it

also to the original poster, i bet if you talked to sporeworks they might be able to supplement the bag for your specific needs


--------------------
We were sent to hell for eternity :hellfire: Ø:omgawesome:h®
We play on earth to pass the time :foreheadslap:

Over-population the root of all Evil-brings the Elites Closer to the gates.

Extras: Filter Print Post Top
Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: makaveli8x8]
    #7923262 -

Gourmet - Your thread was most interesting and most helpful. It's too bad the presentation didn't work out for the best, I bet it would have been badass. It is good to hear that it fruits well at room temp. That will make things much easier. I went ahead and ordered the sawdust kits from sporeworks. I also talked to Workman and just sent an email regarding the pH of these bags. Even if growth takes a bit longer in the sawdust, I assume I can just dunk in some pH 3.5 sodium phosphate buffer to get things going after opening the bag.

makaveli - I will probably do that if growth is too slow. Or I can just make up my own mix and autoclave it here if I can convince someone it would be easier. As of now this is sort of a side project so we're not looking to put too much time and effort into it.

buddha - I don't know wtf you are doing wasting time in my thread, but please stop taking up space unless you are contributing to the discussion.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: GlowMe]
    #7926032 -

GlowMe, I have no idea if sodium phosphate would support fungal growth or if there is some issue with the sodium ions. I cannot recommend it if I have not tried it.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: GourmetMushroom]
    #7929396 -

It would likely be very dilute, ~25mM (.025M) so I doubt it would have any ill effects, but this is only one out of several possible buffers I could use. In fact I'm not even sure if a sodium phosphate solution would buffer well at that low of a pH. I will have to check some tables. From the advice in this thread I might think about doing a citrate-citric acid buffer.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: GlowMe]
    #7929496 -

Or, find some sulfur powder. That will lower pH safely and help to keep it low.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: RogerRabbit]
    #7931214 -

It's not terribly hard to throw some sawdust into a vessel or autoclave bag. I would suggest doing at least a couple cultures using different methods. It's a lot better sounding to cite a woodlover cultivation paper technique with modifications rather than saying you used a commercial kit IMHO.

As far as buffers I don't think you'll run into problems with any of the commonly used ones in the biosciences.

You might actually not want it to be buffered so that you can let it "charge up" a bit and then hit it with a lower pH shortly before you do your extraction/analysis or whatever.


-FF

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: fastfred]
    #7933811 -

did anyone every get prints from these? I didnt see it on sporeworks
site I would love to try this BL mushroom ..

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: GourmetMushroom]
    #7934276 -

Hi, been observing this thread with interest :smile: A thought on phosphate - ammonium phosphate avoids introducing sodium ions, and buffers at a slightly lower pH. Maybe you don't want the extra nitrogen though.

Btw, with european stipticus only the mycelium glows (apparently). I'd love to see some of them little brackets glowing. Here's to the success of the project.


--------------------
Be joyful. This could be the only chance you get.

All of the above posts are the fevered imaginings of a deluded mind, itself entirely the fictional creation of a somewhat peculiar author with a bizarre sense of humour.

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Re: Bioluminescent Fungi - Novel Luciferin Isolation + Structural Determination [Re: Mr E Guest]
    #7954797 -

The package from SporeWorks came yesterday. I inoculated the four bags and they are currently incubating at room temp in the dark (as chosen from that paper) in an unused and empty lab room. I will update with any progress on the project.

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