Bioluminescence in fungi

[Kydelic]

I know this is a pretty basic question and that bioluminescence genes
can be inserted into various organisms as markers and for overall
bioluminescence,
but how complicated is this process and what does it entail? What
sorts of
life can this be used on? I'm particularly interested in seeing
this done to various fungi, including molds and mushrooms.

[Aaron Hicks]

That's a mighty big question; sticking genes into plants is very different from animals- and even then, there's birds versus cats versus dogs versus fish. Then there's transient versus stable transformation.

Transgenic fungi aren't what you'd call a big market. There's less of a call for it than with other organisms. There are tens of fungi that are already luminescent, too. Slime molds have been done; here's an old paper on the subject:

Gene. 1995 Nov 7;165(1):127-30. Links

Green fluorescent protein production in the cellular slime molds Polysphondylium pallidum and Dictyostelium discoideum.

Fey P, Compton K, Cox EC.

Department of Molecular Biology, Princeton University, NJ 08544, USA.

The green fluorescent protein-encoding gene from Aequorea victoria has been cloned into several different transforming vectors and expressed in the cellular slime molds, Polysphondylium pallidum and Dictyostelium discoideum. We find that the protein is stable and non-toxic in both species, can be easily visualized in living and fixed specimens, and can be used to purify rare cells by fluorescence-activated cell sorting (FACS).

-AJ

[Jake]

You're getting GPF and fluorescence confused with bioluminescence
(actual glowing). Transforming something with a GFP gene is about as
easy as it gets. Bacteria, plants and fungi are simple to transform.
Bacteria can usually be done with a simple electroporation and all
three can be transformed with agrobacterium.

I'm a bit curious myself about how hard it would be to get actual
bioluminescence working. I haven't read much about it being done.


-Jake

[Kyle Stratis]

That's right, I was confusing the two...I am initially interested in getting
work done with GFP in fungi (especially various mushroom species
as decoration), and, eventually, when I get more knowledge and skills
actual bioluminescence.

[Jake]

Well the GFP is pretty simple. There are a couple papers out there on
fungi if I remember right.

You might also want to check out pCAMBIA. They have a funGUS vector.
It's a eukaryotic beta-glucuronidase discovered in fungi. Basically it
makes your tissue blue where it's expressed. There are some
advantages over GFP in a lot of cases.
http://www.cambia.org/daisy/cambialabs/3709.html

Maybe someone can jump in here about bioluminescence? I see a lot of
research and products using luciferase, but you have to buy the spendy
luciferin to go along with it. Has the luciferin pathway not been
sequenced? Obviously the biotech companies are making it somehow. It
sure would be nice if there was a luciferin cassete vector out there.
A quick search shows a few steps in synthesis and a "luciferin
regenerating enzyme" that's been sequenced.


-Jake

[Cory Tobin]

> You might also want to check out pCAMBIA.  They have a funGUS vector.
> It's a eukaryotic beta-glucuronidase discovered in fungi. Basically it
> makes your tissue blue where it's expressed.  There are some
> advantages over GFP in a lot of cases.
> http://www.cambia.org/daisy/cambialabs/3709.html

If the goal is to make a funny-colored fungus that is alive, then GUS
won't work. The way a GUS assay works is you dissect and fix the
tissue and then add a chemical like X-Gluc that, when cleaved by the
enzyme, makes a chemical that is blue. Just expressing
beta-glucuronidase doesn't make blue tissue.

> Maybe someone can jump in here about bioluminescence?  I see a lot of
> research and products using luciferase, but you have to buy the spendy
> luciferin to go along with it.  Has the luciferin pathway not been
> sequenced? Obviously the biotech companies are making it somehow.  It
> sure would be nice if there was a luciferin cassete vector out there.
> A quick search shows a few steps in synthesis and a "luciferin
> regenerating enzyme" that's been sequenced.

Luciferase is similar to GUS in that expressing the protein in a
fungus isn't going to make it fluoresce. The protein catalyzes the
oxidization of the chemical luciferin which releases light. So you
would have to bathe the fungus in luciferin to make it glow. But
obviously fireflies glow without being bathed in luciferin. In this
case they synthesize the luciferin themselves. So it could be
possible to engineer a system that synthesizes the luciferin as well
as the luciferase. I think there was a discussion about doing this in
brewing yeast a while back, although searching the google group for
luciferase doesn't turn up any results. Or maybe I'm just computer
stupid.

-Cory

[Jake]

After a quick search it seems that the luciferin pathway isn't well
understood in any organisim I could find. What a bummer! You would
think scientists would be all over this. I've seen plenty of popular
articles (pop sci, etc.) on people trying to make bio-lights and
commercialize this.

I think it would be pretty important to figure this out.

All sorts of things are bioluminescent from bacteria and plankton to
fungi to fish. It doesn't seem that it would be that hard to mutate
bacteria using insertional mutagenesis isolate non-glowing bacteria,
then recover the genes using reverse PCR or plasmid rescue.

From wikipedia...

Proposed applications...
Glowing trees to line highways to save government electricity bills
Christmas trees that do not need lights, reducing danger from
electrical fires
Agricultural crops and domestic plants that luminesce when they need
watering
New methods for detecting bacterial contamination of meats and other
foods
Bio-identifiers for escaped convicts and mental patients
Detecting bacterial species in suspicious corpses
Novelty pets that bioluminesce (rabbits, mice, fish etc.)
[...]
The in vivo synthesis of firefly luciferin is not completely
understood. Only the final step of the enzymatic pathway has been
studied, which is the condensation reaction of D-cysteine with 2-
cyano-6-hydroxybenzothiazole, and is the same reaction used to produce
the compound synthetically.


-Jake

[rallodc45]

There is a paper on pubmed: (http://www.pubmedcentral.nih.gov/
articlerender.fcgi?artid=365129) regarding luciferase. If I am reading
it correctly (the abstract), they were able to express the gene in
monkey cells. I haven't read the whole paper, but it seems within the
realm of possibility.

[Ben Gadoua]

Rallodoc;

  What they were discussing was that luciferase on it's own is not enough, it needs the substrate, luciferin, to produce luminescence. Luciferase works by enzymatically catalyzing the oxidation of luciferin. There are many, many, viral and plasmid vectors to induce the production of the luciferase enzyme, however there are no vectors currently published that allow you to make luciferin. Luciferin is quite expensive and you need to saturate the organism to make it luminese.

Ben

[Cory Tobin]

I just found the thread where this kind of thing was discussed previously:
http://groups.google.com/group/diybio/browse_thread/thread/6be71211d0082842/dd7d1ba08c42388f?#dd7d1ba08c42388f

It's a fairly long discussion but one thing to note was a reference to
a paper where a self-sufficient bioluminescence system was created in
yeast. The transgenic yeast produced both the enzyme and the
substrate.
http://aem.asm.org/cgi/reprint/71/8/4455.pdf

I think the conclusion from the discussion was that making a
luminescent organism would be much more difficult than a fluorescent
organism, but it is possible. That article linked to above might be a
good place to start.

-Cory

[Jake]

Thanks for catching my unclarity about GUS. Kind of the same problem
I guess, but it probably wouldn't be too hard to produce the x-gal or
whatever in vivo.

Anyhow, after looking for awhile there IS a luciferin that's been
discovered.

Check out US patent # 5741668

Coelenterazine can be produced transgenically. Interestingly it's a
modified GFP that actually becomes coelenterazine. No complex
biosynthetic pathway at all. They were amazed to discover that a GFP
with one different residue magically becomes coelenterazine (a
luciferin) after a non-specific biotransformation.

BTW Cory the paper you referenced requires aldehydes to work and uses
bacterial lux genes. Not really true bioluminesce except in certain
aldehyde producing bacteria.


-Jake

[Tom Knight]

On Aug 17, 2009, at 11:35 PM, Jake wrote:

>
> BTW Cory the paper you referenced requires aldehydes to work and uses
> bacterial lux genes. Not really true bioluminesce except in certain
> aldehyde producing bacteria.
>

That's not right. The complete bacterial system produces aldehyde by
reducing fatty acids. The prototype system is the Vibrio fischeri Lux
system, Genbank AF170104, with genes LuxCDEF creating the aldehyde
synthesis pathway, and LuxAB being the luciferase. I don't know if
this can be expressed in eukaryotes, but I don't see any particular
reason why not.

[Jake]

Thanks for the update. Last I read they were still pumping plants
full of aldehydes to get the bioluminesce in transgenic tobacco.

-Jake

[Tom Randall]

On Aug 14, 11:41 pm, Kyle Stratis <telehorse....@gmail.com> wrote:
> That's right, I was confusing the two...I am initially interested in getting
> work done with GFP in fungi (especially various mushroom species
> as decoration), and, eventually, when I get more knowledge and skills
> actual bioluminescence.

Here are some available reporter genes for fungi: http://www.fgsc.net/plasmid/reporter.html
However, these are for ascomycetes, I am not sure if their promoters
would function in a mushroom (basidiomycete) but there should be
somebody out there who has built a functional GFP reporter gene for a
basidiomycete, may or may not be available. Maybe search PubMed. More
specifically, you might also need a promoter active in the fruiting
body of the shroom of interest so you might have to construct it
yourself eventually. At least the coding sequence of these GFPs would
be optimized for fungi and would likely work in a basidiomycete also,
but that could be another issue.

[Christina Agapakis]

Here is a paper with quite detailed methods and references on genetic
engineering of mushroom forming fungi.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1456251

[Jake]

Can't seem to dig up the paper at the moment, but in the GFP in
agaricus (button mushroom) paper I read they found they needed to add
an intron to get good expression. I also recall some promoter papers
on basidiomycetes, so the research is there.

-Jake

[Nathan McCorkle]

luminescent mushrooms already exist.... check out the comments here:
http://www.mykoweb.com/CAF/species/Omphalotus_olivascens.html

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Nathan McCorkle]

sorry for not trimming,,,

wiki says it's got luciferase:
http://en.wikipedia.org/wiki/Omphalotus_olearius

[Nathan McCorkle]

this is also a great site for fungi info
http://botit.botany.wisc.edu/toms_fungi/oct97.html

I will see if I can find some spores somewhere

[Jason Kelly]

> I will see if I can find some spores somewhere

one stop shop for spawn:
http://www.fungi.com/

founded by this guy --

good talk:
http://www.ted.com/talks/paul_stamets_on_6_ways_mushrooms_can_save_the_world.html

good book:
http://www.amazon.com/Mycelium-Running-Mushrooms-Help-World/dp/1580085792/ref=sr_1_1?ie=UTF8&s=books&qid=1250662265&sr=8-1

thanks,
jason

[Nathan McCorkle]

On Wed, Aug 19, 2009 at 2:17 AM, Jason Kelly <jrk...@gmail.com> wrote:

> I will see if I can find some spores somewhere

one stop shop for spawn:
http://www.fungi.com/

I will search there, already  tried sporeworks.com

founded by this guy --

good talk:
http://www.ted.com/talks/paul_stamets_on_6_ways_mushrooms_can_save_the_world.html

good book:
http://www.amazon.com/Mycelium-Running-Mushrooms-Help-World/dp/1580085792/ref=sr_1_1?ie=UTF8&s=books&qid=1250662265&sr=8-1

Yeah Stamets is the  man, you can torrent that talk to watch offline... and I've had that book since publishing, some awesome ideas on myco-based water filters and such...

thanks,
jason

On Wed, Aug 19, 2009 at 2:08 AM, Nathan McCorkle<nmz...@gmail.com> wrote:
> this is also a great site for fungi info
> http://botit.botany.wisc.edu/toms_fungi/oct97.html
>
> I will see if I can find some spores somewhere
>
>
> >
>

[Kyle Stratis]

Another glowing species, Panellus stipticus, is available
at many of the typical spore vendors. Here is a thread on
the shroomery with some pictures of glowing mycelium.
Apparently the fruiting bodies also glow.

http://www.shroomery.org/forums/showflat.php/Number/10861462

Spore works sells spore syringes:
http://sporeworks.com/store/product.php?productid=16144

[Eugen Leitl]

So who wrote what? See below, it's impossible to figure out.

Why do you bother writing at all if nobody will read it?

On Wed, Aug 19, 2009 at 01:51:14PM -0400, Kyle Stratis wrote:
>
> On Wed, Aug 19, 2009 at 2:37 AM, Nathan McCorkle <[1]nmz...@gmail.com>
> wrote:
>
> On Wed, Aug 19, 2009 at 2:17 AM, Jason Kelly <[2]jrk...@gmail.com>

> wrote:
>
> > I will see if I can find some spores somewhere
>
> one stop shop for spawn:

> [3]http://www.fungi.com/
>
> I will search there, already tried [4]sporeworks.com

>
> founded by this guy --
> good talk:

> [5]http://www.ted.com/talks/paul_stamets_on_6_ways_mushrooms_can_sa
> ve_the_world.html
> good book:
> [6]http://www.amazon.com/Mycelium-Running-Mushrooms-Help-World/dp/1
> 580085792/ref=sr_1_1?ie=UTF8&s=books&qid=1250662265&sr=8-1

>
> Yeah Stamets is the man, you can torrent that talk to watch
> offline... and I've had that book since publishing, some awesome ideas
> on myco-based water filters and such...
>
> thanks,
> jason
>

> On Wed, Aug 19, 2009 at 2:08 AM, Nathan McCorkle<[7]nmz...@gmail.com>

> wrote:
> > this is also a great site for fungi info

> > [8]http://botit.botany.wisc.edu/toms_fungi/oct97.html

> >
> > I will see if I can find some spores somewhere
> >
> >
> > >
> >
>
> --
>
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics
>
> Another glowing species, Panellus stipticus, is available
> at many of the typical spore vendors. Here is a thread on
> the shroomery with some pictures of glowing mycelium.
> Apparently the fruiting bodies also glow.

> [9]http://www.shroomery.org/forums/showflat.php/Number/10861462

>
> Spore works sells spore syringes:

> [10]http://sporeworks.com/store/product.php?productid=16144
> >
> References
>
> 1. mailto:nmz...@gmail.com
> 2. mailto:jrk...@gmail.com
> 3. http://www.fungi.com/
> 4. http://sporeworks.com/
> 5. http://www.ted.com/talks/paul_stamets_on_6_ways_mushrooms_can_save_the_world.html
> 6. http://www.amazon.com/Mycelium-Running-Mushrooms-Help-World/dp/1580085792/ref=sr_1_1?ie=UTF8&s=books&qid=1250662265&sr=8-1
> 7. mailto:nmz...@gmail.com
> 8. http://botit.botany.wisc.edu/toms_fungi/oct97.html
> 9. http://www.shroomery.org/forums/showflat.php/Number/10861462
> 10. http://sporeworks.com/store/product.php?productid=16144
--
Eugen* Leitl <a href="http://leitl.org">leitl</a> http://leitl.org
______________________________________________________________
ICBM: 48.07100, 11.36820 http://www.ativel.com http://postbiota.org
8B29F6BE: 099D 78BA 2FD3 B014 B08A 7779 75B0 2443 8B29 F6BE

[Tito Jankowski]

Cool!

Mycelium Running: How mushrooms can help save the world

The first 100 pages of the 356 page book are available for free on Google to see if you're interested in the full book.

http://books.google.com/books?id=NPI8_-omzvsC&dq=Mycelium+Running&printsec=frontcover&source=bn&hl=en&ei=9M6MStKyPI_6sQOd0fTcCQ&sa=X&oi=book_result&ct=result&resnum=7#v=onepage&q=&f=false

Tito

[Kyle Stratis]

On Wed, Aug 19, 2009 at 4:11 PM, Eugen Leitl <eu...@leitl.org> wrote:

So who wrote what? See below, it's impossible to figure out.

Why do you bother writing at all if nobody will read it?

Perhaps you need better email software, I know who wrote what
and I think it is fairly obvious who wrote what, and what I was replying to.
I'd consider an upgrade from mail.

[Kyle Stratis]

Another point I'd like to address is that different mail clients
handle quoting very differently. Some use >, others use
rendered lines (like gmail)...some try, craptastically, to convert them
which makes a mess.

[Bryan Bishop]

On Thu, Aug 20, 2009 at 8:08 PM, Kyle Stratis wrote:
> Another point I'd like to address is that different mail clients
> handle quoting very differently. Some use >, others use
> rendered lines (like gmail)...some try, craptastically, to convert them
> which makes a mess.

Sorry, you're lying about gmail. That's completely configurable under
the gmail web interface. The quoting is also configurable under most
email clients. Maybe it's you who needs to upgrade to meet list
standards?

- Bryan
http://heybryan.org/
1 512 203 0507

[Kyle Stratis]

The last time Eugen said something about this I asked what the standards were
and no one said anything changing the quotes. I didn't know we had to change
how our clients handled quoting (or even that gmail was configurable for that)
for the list standards. Again, why isn't there a file for list standards, since they
seem to cause so much controversy for the new user?

[Bryan Bishop]

On Thu, Aug 20, 2009 at 8:18 PM, Kyle Stratis wrote:

> On Thu, Aug 20, 2009 at 9:12 PM, Bryan Bishop wrote:
>> On Thu, Aug 20, 2009 at 8:08 PM, Kyle Stratis wrote:
>> > Another point I'd like to address is that different mail clients
>> > handle quoting very differently. Some use >, others use
>> > rendered lines (like gmail)...some try, craptastically, to convert them
>> > which makes a mess.
>>
>> Sorry, you're lying about gmail. That's completely configurable under
>> the gmail web interface. The quoting is also configurable under most
>> email clients. Maybe it's you who needs to upgrade to meet list
>> standards?

There is a file about list standards up on the web. It's in the FAQ,
but it's also been discussed previously.

http://openwetware.org/wiki/DIYbio/FAQ

Unfortunately diybio.org doesn't link to the FAQ so I can see why this
might be a problem.

[Kyle Stratis]

Yeah, I found the list from the DIYBio site and since the
discussion about the FAQ (which I think had more to do with
the purpose of skdb and some other things). I searched around
on how to change the quoting, I'm not sure if other gmail users
looked around for it, but simply converting to plaintext in the reply
body will do it. Back to the regularly scheduled discussion