Engineering Plant Bioluminescence (was Re: experimentation)

[Cathal Garvey]

Brilliant though! That's something to work with at least. There are many things that could have been affecting things to slow down light emission. Codon optimization, protein separation, bottlenecking at any stage.. perhaps by codon optimizing and directly chaining proteins we could get it working? I saw linkage at work on a B12-13 pathway once, it was well over tenfold improvement by my recollection.

Time to read that paper!

On 15 Mar 2011 21:45, "Cory Tobin" <cory....@gmail.com> wrote:

> As to bioluminescent plants, from my understanding they have never yet been
> made to glow indepen...

Sorry to interrupt the conversation on frankenstein experiments. One
small correction about the plants.  There was one case where someone
built the entire luciferin synthesis pathway in plants.
http://dx.plos.org/10.1371/journal.pone.0015461 (PLoS One, open
access)  Unfortunately the amount of luciferin it produced was so low
they could only see the autoluminescence with a five minute exposure
in the dark.  So it wasn't visually stunning :[


-cory

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[Cathal Garvey]

Yea, to summarise for others:
They were taking the operon for luciferase/in from a wild gram
negative and inserting it into the Chloroplasts in order to determine
whether it would work due to the fact that Chloroplasts are basically
just highly specialised bacterial symbiotes. As it happens, it
worked, but the results were (as Cory already said) unspectacular.

Were you to engineer this for optimal expression in Chloroplasts, or
in plant cells with compartmentalisation in the chloroplast if needed
for Luciferin synthesis, you'd do a lot better. :)

On Tuesday, 15 March 2011, Cathal Garvey <cathal...@gmail.com> wrote:
> Brilliant though! That's something to work with at least. There are many things that could have been affecting things to slow down light emission. Codon optimization, protein separation, bottlenecking at any stage.. perhaps by codon optimizing and directly chaining proteins we could get it working? I saw linkage at work on a B12-13 pathway once, it was well over tenfold improvement by my recollection.
>
> Time to read that paper!
> On 15 Mar 2011 21:45, "Cory Tobin" <cory....@gmail.com> wrote:
>
>> As to bioluminescent plants, from my understanding they have never yet been

>> made to glow indepen...Sorry to interrupt the conversation on frankenstein experiments. One

> small correction about the plants.  There was one case where someone
> built the entire luciferin synthesis pathway in plants.
> http://dx.plos.org/10.1371/journal.pone.0015461 (PLoS One, open
> access)  Unfortunately the amount of luciferin it produced was so low
> they could only see the autoluminescence with a five minute exposure
> in the dark.  So it wasn't visually stunning :[
>
>
> -cory
>
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>

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[Cathal Garvey]

Yea, to summarise for others:
They were taking the operon for luciferase/in from a wild gram
negative and inserting it into the Chloroplasts in order to determine
whether it would work due to the fact that Chloroplasts are basically

bacteria. It works, but not very well. Codon optimization plus more
appropriate regulatory sequences please!

On Tuesday, 15 March 2011, Cathal Garvey <cathal...@gmail.com> wrote:

> Brilliant though! That's something to work with at least. There are many things that could have been affecting things to slow down light emission. Codon optimization, protein separation, bottlenecking at any stage.. perhaps by codon optimizing and directly chaining proteins we could get it working? I saw linkage at work on a B12-13 pathway once, it was well over tenfold improvement by my recollection.
>
> Time to read that paper!
> On 15 Mar 2011 21:45, "Cory Tobin" <cory....@gmail.com> wrote:
>
>> As to bioluminescent plants, from my understanding they have never yet been

>> made to glow indepen...Sorry to interrupt the conversation on frankenstein experiments. One

> small correction about the plants.  There was one case where someone
> built the entire luciferin synthesis pathway in plants.
> http://dx.plos.org/10.1371/journal.pone.0015461 (PLoS One, open
> access)  Unfortunately the amount of luciferin it produced was so low
> they could only see the autoluminescence with a five minute exposure
> in the dark.  So it wasn't visually stunning :[
>
>
> -cory
>
> --
> You received this message because you are subscribed to the Google Groups "DIYbio" group.
> To post to...
>

[Mega]

One plasmid of the pGreen plasmids (agrobacterium) already carries a firefly luciferase.

I may want to try this as an (quite easy) next step. Just put the two plasmids into agrobacterium and put it on a leave.
I got connected to a small tissue culturing lab, so they would help hopefully.


If you add the beetle luciferin, will it glow so strong to be seen with your naked eye??

 
 

[Cathal Garvey]

Yes, but as far as I'm aware there are no genetic systems available to
*make* the luciferin that fireflies use. It's different to bacterial
luciferin, so you can't mix/match from the two systems.

While firefly luciferin is great for visibility (because you can add the
luciferin aldehyde in bulk), you have to buy the luciferin, and it's
really expensive and prone to degradation when out of the freezer.

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[Andreas Sturm]

Yeah clearly, but this time I'll do it for demonstration / educational purpose in cooperation with a tissue culture lab.

We'd buy some luciferin... How  much will we need??

2012/8/9 Cathal Garvey <cathal...@gmail.com>

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[Jeswin]

On Thu, Aug 9, 2012 at 6:51 AM, Cathal Garvey <cathal...@gmail.com> wrote:
> While firefly luciferin is great for visibility (because you can add the
> luciferin aldehyde in bulk), you have to buy the luciferin, and it's
> really expensive and prone to degradation when out of the freezer.
>

Are there any alternatives to the luciferase system?

[Mega]

There is the bacterial lux sytem which contains all the genes needed to produce light. but as the name says only bacteria express it (operon)

Is there a way to find out how much luciferin we'll need to buy?

[Cathal Garvey]

Honestly, depends on how much you plan to "burn". Really, the limiting
factor won't be the amount of Luciferin, but the expression level of
your luciferase. If you can get it expressing at truly epic levels,
it'll be quite visible, limited by oxygen concentration more than
luciferin. Look up that glowing tobacco plant picture; that was done
with Firefly Luciferase and added luciferin.

If you're planning to go down this route, look into luciferin
regeneration systems. Although pathways to make the luciferin from
scratch are either unknown or unpublished, there are systems that will
recycle used luciferin to some extent, significantly enhancing and
prolonging the glow.

Fun fact; there's at least one species of (Atlantic?) fish that has a
Luciferase enzyme complex, but seemingly doesn't make its own Luciferin.
It only glows in the parts of the ocean where its diet includes another
fish that makes luficerin. Thereafter, it glows all the time due to
recycling of ingested luciferin. Nature!

On 10/08/12 22:03, Mega wrote:
> There is the bacterial lux sytem which contains all the genes needed to produce light. but as the name says only bacteria express it (operon)
>
>
> Is there a way to find out how much luciferin we'll need to buy?
>

[Jeswin]

On Fri, Aug 10, 2012 at 5:03 PM, Mega <masters...@gmail.com> wrote:
> There is the bacterial lux sytem which contains all the genes needed to produce light. but as the name says only bacteria express it (operon)
>

No, I was wondering if there is another bioluminescene system other
than lux. Maybe something that doesn't use luciferin?

[Andreas Sturm]

Well, there is aequorin+Coelenterazin. But even here you have to add the latter substance.

[Cathal Garvey]

"Luciferin" and "Luciferase" are generalised terms for the components of
any system that primarily emits light.

So, if you discovered examplase, an enzyme that digests placeholderin (a
random substrate) to emit light, then examplase would be A luciferase,
and placeholderin would be A luciferin.

Hence much confusion; luciferase systems often have no relationship to
one another at all, except that they emit light. The common nomenclature
is meaningless.

[Jeswin]

On Wed, Aug 15, 2012 at 11:53 AM, Cathal Garvey <cathal...@gmail.com> wrote:
> "Luciferin" and "Luciferase" are generalised terms for the components of
> any system that primarily emits light.
>

Kinda misleading statement. I looked up luciferin now and it is a
class of molecules that produce light energy when oxidized.

> Hence much confusion; luciferase systems often have no relationship to
> one another at all, except that they emit light. The common nomenclature
> is meaningless.
>

So is it possible to find the cheapest luciferin compound available
and use that particular system?

I wonder how hard it is to extract Dinoflagellate luciferin coumpounds?

[Andreas Sturm]

>So is it possible to find the cheapest luciferin compound available
>and use that particular system?

In theory yes. But the luciferase has to be available for your target species.
Bacterial Lux A + B are in an operon, they have to be cut out (+ yeast promotor, etc.) when inserting e.g. in yeast.

[Cathal Garvey]

> I wonder how hard it is to extract Dinoflagellate luciferin

> compounds?

I don't know about extraction, but IIRC the Dinoflagellate system is a
calcium-sensitive system, which makes it less "turn-key" in transgenic
systems. That is, your target cell might have abundant calcium in cell
cytoplasm, saturating the luciferase, perhaps disrupting the cycle of
binding-digestion-release-recyling. More likely, it'll just mean it
burns luciferin at maximum rate though, to be fair.

Honestly, bacterial luciferase/in is a pretty good system as-is, and
with a bit of work you could either find Tetradecanal (the bacterial
luciferin) for sale directly, or buy Tetradecanol (more commonly
available as a food additive) and try to chemically alter it to an
aldehyde manually.

[Nathan McCorkle]

On Wed, Aug 15, 2012 at 6:30 PM, Sebastian <scoc...@gmail.com> wrote:
> I worked in the lab where the experiment was done. The guy, Alex, started a
> company and patented the methods for producing the plant. I am very
> interested in finding alternatives to their procedure since I am in the
> plant biotech business myself. If you do, however decided to try it just
> make sure you dont infringe upon his IP in any way. Happy, and safe,
> hacking!

Sebastian, did your friend patent the DNA sequences or the arrangement of them?

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[Patrik D'haeseleer]

Dinoflagellates actually use a modified version of their chlorophyll as luciferin, but if I remember correctly, it oxidizes rapidly in air (without producing a photon, unfortunately).

The genes for turning the chlorophyll into luciferin are unknown, and there is no dinoflagellate reference genome available yet (they have genomes that are much bigger than ours). We're hoping to chase after some of those genes at BioCurious, since we've been doing lots of growth experiments with dinoflagellates, but this would admittedly be quite an ambitious project.

As far as I know, the bacterial bioluminescence system is the only one where the luciferin biosynthesis pathway is known. So unless you're willing to buy and add luciferin exogenously, you're probably stuck with the bacterial system. The fact that we know the entire pathway also means this is a great target for metabolic engineering. I've seen precious little about trying to optimize light output by balancing the different enzymes involved. I wouldn't be surprised if you could increase light output tenfold even in E. coli.

[Sebastian Cocioba]

From what I know, you cannot patent a natural phenomenon. He patented the method for transformation and the resulting plants which arise from said transformation. Here is the patent in all its lengthy glory:  http://www.sumobrain.com/patents/wipo/Autoluminescent-plants-including-bacterial-lux/WO2011106001.html If u have questions, keep em coming. Id love to help. Btw I run a small micropropagation lab and can lend my services to the DIY community for a very reasonable price (normally cost of materials + 15%) and will give discounts if the project is cool and awesome and whatnot :P Happy Hacking!

[Andreas Sturm]

So now its forbidden to cut out the bacterial lux genes and put into plant chloroplasts? ^^

Cannot be thus. God holds the patent of bioluminescence. Maybe just the way of getting it into chloroplasts though? But particle bombardement is a standard lab technique, why could one patent that?

 

2012/8/16 Sebastian Cocioba <scoc...@gmail.com>

[Andreas Sturm]

Anyone knows where to get the cheapest beetle luciferin??

[Sebastian Cocioba]

Pvib from carolina.com is fairly cheap although it might lack some of the other genes like luxG and flavins that enhance the glow. Pvib is from vibrio fischeri or the victoria jelly fish but either way its made into a bacterial vector. You might need some plastic vector promotors and upstream regulators. Ill keep you guys posted on anything I find as a nice alternative. You will hear it first :)

On Thursday, August 16, 2012, Andreas Sturm wrote:

Anyone knows where to get the cheapest beetle luciferin??

--

[Sebastian S. Cocioba]

It was the specific way he did it that is patented against profit. No one is stopping you from replicating the experiment for educational purposes.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

[in Quantum entanglement]

That needs a robust design to conduct experiment with.luxgene

[Sebastian Cocioba]

If you follow what has been done already you can have an idea of the equipment, techniques, and materials required to repeat let alone modify their experiment. Now if someone can make a Piezoelectric Oligonucleotide Synthesis Machine and/or an Open Source Gene Gun...much DIY progress can be made. Hint hint to all those handy electrical engineers out there. OpenPCR is making a killing and the device is simple. There are plans out there...

[Andreas Sturm]

Yeah, but I was looking for beetle luciferin. Gonna transfect a plant tissue culture with agrobacterium-pGreenII 0049, and that one needs luciferin...

2012/8/16 Sebastian Cocioba <scoc...@gmail.com>

[Patrik D'haeseleer]

P212121 has some great prices on luciferin (and lots of other things!).

http://store.p212121.com/search.php?search_query=luciferin&x=0&y=0

We had originally decided not to work with firefly luciferase at BioCurious because of the cost, but P212121 has it at something like 1/3 the best price we were able to find elsewhere.

Patrik

[Andreas Sturm]

Thanks a lot...


What about the plasmid, I was to get pGreenII 0049 (Luciferase included). It costs some 40 british pounds (i think 60 $)

Is there another system you can buy, maybe cheaper?? And also, maybe not a binary system, but where one plasmid carries all genes?

2012/8/17 Patrik D'haeseleer <pat...@gmail.com>

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[Andreas Sturm]

They  (from pGreenII website) are not answering... Did a request last Sunday.

(Ok, they sent an email that they are busy and answer e-mails only on friday afternoons. But now it's saturday... )



Are there alternatives to get a Luc+ agrobacterium?? We need to order one kit ASAP  to get it done. One week waiting delays our project by one week.

[Nathan McCorkle]

can you find one with the bacterial luciferase system? so you don't
have to add luciferin?

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[Andreas Sturm]

No, can't find one.... Very unfortunately....

You just would have to add Decanal (cheap aldehyde) and flavine.



If someone accidentally knows one, would be very grateful ;)

2012/8/18 Nathan McCorkle <nmz...@gmail.com>

[Jordan Miller]

the whole bacterial lux operon is supposedly available as an iGEM biobrick. 

jordan

[Dakota Hamill]

Spent most of yesterday reading all through their lab notebooks listed online.

http://2010.igem.org/Team:Cambridge/Bioluminescence/G28

They did a neat project, all on the lux operon, and pretty sure it's available under the pBAD promoter.  

[Sebastian Cocioba]

Quick questions:

Are you guys attempting full blown autoluminescence in plants?

Have you tried chloroplast transformation since it still contains prokaryotic gene expression tools which can help in utilizing bacterial lux genes and actually generate light?

Where is your target insertion region (nuclear, mitochondria, chloroplast, etc)?

What are you using to do the transformation? Agro? Gene Gun? Protoplast madness?

Being a non-iGEM independent researcher trying to find alternatives to current autoluminescent methods, I am curious as to what exactly your approach is. Thanks and as always happy hacking!

--

[Andreas Sturm]

We (me and a tissue culture lab) wanna try to get a plant glowing.

Agrobacterium - firefly luciferase would be the easiest first step, with externally added luciferin.



It's a pitty, but the bacterial lux system is an operon, no chance for plants to express.

Chloroplast transformation will be doable, but cloning the vector (flanking site right PCR - plantPromoter PCR - KanamycinR PCR - xxx (Lux) xxx  -  flanking site left PCR. A lot of PCR steps though...

[Sebastian Cocioba]

Adding external luciferin topically? Will that pass through the cuticle? 

--

[Nathan McCorkle]

chloroplasts can express polycistronic elements I believe (google it),
owing to their bacterial heritage... the bacterial operon should be
fine

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[Sebastian Cocioba]

I posted a link a while ago on THE article about successful autoluminescent plants without the need for exogenous luciferin. Its been done through chloroplast manipulation and works but is very dim. I'm not sure its worth the effort to do the luciferin topical application route besides for the educational experience since it will make an obvious dependence on external chems.

[Nathan McCorkle]

On Sun, Aug 19, 2012 at 3:08 PM, Sebastian Cocioba <scoc...@gmail.com> wrote:
> I posted a link a while ago on THE article about successful autoluminescent
> plants without the need for exogenous luciferin. Its been done through
> chloroplast manipulation and works but is very dim. I'm not sure its worth
> the effort to do the luciferin topical application route besides for the
> educational experience since it will make an obvious dependence on external
> chems.

Do you mean the patent you posted? It was so long I got bored and
stopped reading (I think that must be the point of patents, to bore
you out of copying someone else's work)

It would be nice if you could abbreviate here, and tell us what method
NOT to follow, since your friend patented it.

[Sebastian Cocioba]

Sounds good. Ill get to work on that now and post it in a nice summary and/or pdf report upon request since I need to sift through the patent myself so that we all can know. Everyone is more than welcome to double check my findings and correct any errors.

[Sebastian Cocioba]

To not waste time, here is a link to the open access article about the world's first autoluminescent plant.  http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015461 

I will still compile a summary of what was done, how it was done, and legal issues regarding the commercial and or educational use of said plants.

[Cathal Garvey]

I believe the Cambridge team did just that simply by chopping the Lux
operon in two, so that they could independently regulate (read:
overexpress) the LuxCDEF genes while keeping the LuxAB genes steady. It
seems nature simply hasn't selected for levels of light production that
please the aesthetics of diurnal apes, but the scope is there if you
want to try.

So, instead of Promoter->LuxA/LuxB/LuxC/LuxD/LuxE... you get:
Promoter->LuxA/LuxB (or possibly a fusion protein LuxAB)
Promoter->LuxC/LuxD/LuxE.. (or possibly a fusion protein LuxCDE)

I would wager that fusing CDE transcriptionally would increase yields of
Luciferin; this has been the case in a few other metabolic engineering
projects I've looked over, and some may remember the (Ukraine?) iGEM
team that used fusions to DNA-binding domains to bring several proteins
in a metabolic production-line together, finding that they got far
higher yields by doing so. Proximity is everything, and a protein fusion
is the dumbest way to bring several proteins into proximity.

Doing the same with LuxAB would give:
Promoter->LuxAB->STOP....Promoter->LuxCDE->STOP.
It's anyone's guess if this is worthwhile; certainly a large protein
like LuxCDE is somewhat more prone to translational stalling than a
series of smaller proteins. They only way to find out, as usual, is to
try. :)

>>> On Wed, Aug 15, 2012 at 11:53 AM, Cathal Garvey <cathal...@gmail.com<javascript:>>

[David Murphy]

Are there any well known model bioluminescent fungi? How practical would it be to take an already bioluminescent fungus and try to ramp up the pathways involved rather than attempting to put the genes involved into something else?

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[Cathal Garvey]

There are several/many, but one is readily available as novelty-spawn
for growing bioluminescent fungi at home. I can't recall the species
name offhand, but I looked up some research on that system some time
ago, asking a similar question.

Turns out, they're not quite sure what causes the bioluminescence in
this case, but they reckon it involves peroxide-powered degradation of
the luciferin outside the cell (I think?). Seemingly the magic is in the
interaction of the luciferin with an otherwise routine cellular process,
which may not work the same way in another species/context.

So, no. You could try hacking that fungus to overproduce luciferin, but
its system doesn't seem to be portable to another species.

[Cathal Garvey]

Wait sorry: Hacking the fungus was exactly what you suggested. Yea, I
think you could, but you'd spend a long time figuring out ways of
engineering that particular fungus, methinks.

On 20/08/12 12:00, David Murphy wrote:

[Andreas Sturm]

I'm not sure its worth the effort to do the luciferin topical application route besides for the educational experience since it will make an obvious dependence on external chems.

But it's a cheap first step. And also simple, if the agrobacterium system is ready-to-use.


The lab I'll be working with wanna do that step-by-step.

2012/8/20 Cathal Garvey <cathal...@gmail.com>

[osazuwa]

This is great stuff.  Cathal, can you please recommend a primer on working with bioluminescence in DIYbio.

[Sebastian S. Cocioba]

Panelus stipticus is the bioglo fungus. It has a circadian luciferin oxidation and is most bright at maturity. There needs to be more research done on the guy. The genome is sequenced but very little is known.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[Meow-Ludo]

On the topic of fungi, there are a ton of bioluminescent ones. A group in Australia have a lab that has discovered numerous of them. They also work with other glowing species. Awesome little lab :)

[Cathal Garvey]

Erm, pVib? :)
Honestly, there are only two standards in bioluminescence in lab
environments: The vibrio operon, and the luciferase gene from fireflies.
The former is more promising because it produces its own luciferin, but
the latter has some powerful niche analytical uses and requires less
DNA/points of failure to get results, provided you're willing to add
your own luciferin (and can ensure it gets into contact with the enzyme).

Honestly, Andreas is the guy to talk to here; he's actually working with
pVib and getting results! I only ever cultured wild bacteria for laughs,
and while that's pretty easy, it involves rotting fish and no actual
molecular-scale biology. Just basic microbiology. And the slight
potential for food poisoning..

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[Andreas Sturm]

Are you sure that panellus stypticus has been sequenced?
Haven't found the sequnce on my web research some time ago.


But soon, I'll have panellus st. growing in my garden (if everything goes right ;) )

I assume, doing a genetic library in yeast and looking for the luminescent strain has been tried?? So pointless to repeat?
 

[Dakota Hamill]

I was just wondering about the genome of that as well, in the context of...say you get a sample of the fungi, how the heck do you pull out the genes you want?  

What was the long arduous way people had to go about pulling out genes before sequencing and bioinformatics?  

 One of my good friends had a fire down near the ocean the other day, and as he was collecting firewood in the dark, he noticed some fungi glowing on some rotten wood.  He mentioned it to me and so I'm going to head right now and see if I can spot some and come back when it's dark.

Multiple species exhibit luminescence, but pulling out the needed genes and putting them into a "bio brick" arrangement would be a hardcore project.  There are some fungi that have symbiotic bacteria living within their mycelium, and I wonder if those cause the glowing in some cases.  I know what I read on the wiki mentioned it is the fungal genes in the above case...maybe transferred a long time ago from bacteria?    

Either way, things that glow are always cool!   I'll go down to the water's edge now and take some pics and see what I can find.

[Sebastian S. Cocioba]

The sequencing is GOING to be sequenced sometime soon according to some colleagues of mine working with fungi, not has been done already. My apologies. Current sequences are just partial 18s ribosomal subunits. I can’t seem to find any work done on isolating the lux genes from pSTIP (should be name of plasmid once you/we do isolate it :P). North American strains, to the best of my knowledge, are the only ones who do glow but there can be some variations between strains that lead to a total lack of glow. Inhibitors are unknown. Heredity of glow genes is known and is a dominant trait. Other than that there is a suspicious silence in the r and d area for p. stipticus.

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[Sebastian S. Cocioba]

Can you snag a sample and grow it on wood chips? J

 

From: diy...@googlegroups.com [mailto:diy...@googlegroups.com] On Behalf Of Dakota Hamill
Sent: Tuesday, August 21, 2012 10:29 AM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Re: Engineering Plant Bioluminescence (was Re: experimentation)

 

I was just wondering about the genome of that as well, in the context of...say you get a sample of the fungi, how the heck do you pull out the genes you want?  

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[Dakota Hamill]

I know for a fact there are a few glowing species near me.  I took this album a year or two ago, and there are some honey mushrooms in there.  

http://www.flickr.com/photos/66689393@N02/sets/72157627694567885/ 

Jack-O-Lantern mushrooms in the woods around my house, whether or not they are displaying fruiting bodies yet, I'll have to check

http://en.wikipedia.org/wiki/Omphalotus_olearius

Honey mushrooms are also around

http://en.wikipedia.org/wiki/Armillaria

here's the honey mushroom pic I took last year

http://www.flickr.com/photos/66689393@N02/6211681325/in/set-72157627694567885

This white fungi...I don't know if this one is around...but I assume it is if it's mistaken for Oyster mushrooms, and oyster mushrooms are around.

http://en.wikipedia.org/wiki/Panellus_stipticus

The only bad  thing is...my camera can't do long exposures.  I'll see what I can grab from the woods though...just waiting for a car to use

If I find any I'll give it a try on some coffee grinds and newspaper/cardboard.  I know oyster mushrooms can grow well on it, so maybe the bitter oytster, paneulls stipticus, will as well.

[Sebastian Cocioba]

http://onlinelibrary.wiley.com/doi/10.1002/bio.1170080403/pdf 

Chemiluminescense in P. Stipticus. Some guys played with the chems found on the shroom and made it glow. Just throwing this out there.

[Dakota Hamill]

Well I went for a walk in the woods, turned out to be a little short because I was being attacked by horse flies and mosquitoes, forgot to bring bugspray.

I found some honey mushrooms, and what I thought were jack-o-lanterns, but on reviewing them online, I think they are golden waxy caps, aka yellow / orange waxy caps.

Here is the whole gallery

http://www.flickr.com/photos/66689393@N02/sets/72157631178779528/

Waxy caps  http://www.bio.brandeis.edu/fieldbio/Edible_Plants_Ramer_Silver_Weizmann/Pages/spp_page_Hygrocybe_flavescens.html

Also found pretty healthy looking ghost plants.  I thought these were some kind of wierd mushroom for my entire childhood...actually up until about a year ago when I finally took one home and dissected it, and noticed it appeared to have anthers, pollen, and a fragrance of a flower.  After some more searching I found out it is indeed a flower, but lacks chlorophyll, and instead gets its nutrients from the mycelium of a fungi...which in turn gets its nutrients from the roots of a hardwood tree.  So...it is parasitic, but doubly removed from a photosynthesizing plant, pretty neat!  I saw tons of really cool things in just 20-30 minutes outside, made me realize I spend way too much time on the computer.    

So, go outside and find some cool things!

http://www.flickr.com/photos/66689393@N02/7832274952/in/set-72157631178779528

and if you want to read about them http://en.wikipedia.org/wiki/Monotropa_uniflora

I set them up for a spore print on paper/cardboard and on a microscope slide, and, though I'm not holding my breath for it, I'll see if I can see any luminescence tonight, and if I'm brave will maybe venture back out into the woods.  

[Sebastian S. Cocioba]

Sounds like quite the adventure! Good luck tonight!

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

[Andreas Sturm]

Awsome!!

2012/8/21 Sebastian S. Cocioba <scoc...@gmail.com>

[Sebastian S. Cocioba]

Not gonna lie, the agar in a cupcake tin is both the most adorable and brilliant biotech hack I have seen in a while :) biobrowniepoints!

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

[Nathan McCorkle]

I've seen those 'ghost plants' before too, and always thought they
were a really weird looking fungi, and wasn't actually sure what it
was... glad to hear the keyword 'ghost plants'!!!

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[Cathal Garvey]

The "old fashioned way" is to perform a cDNA library with a
promoter-armed plasmid; essentially, find some glowing tissue, quickly
extract mRNA from said tissue, then reverse transcribe to cDNA and clone
into a plasmid with an active promoter and transform E.coli or yeast.

Of course, because traits like this will almost certainly require more
than one gene, and fungi don't use operons, any individual gene you find
won't on its own confer glowing traits in this way.

An alternative is to perform whole-chromosome digestion/fractionation
and blunt clone large fragments of DNA into a vector compatible with a
species more likely to recognise P.stipticus promoters, and then just
transform and hope for a glowing strain. If you find one, you sequence
the chromosomal fragment and start guessing which genes encode luciferin
synthases and luciferases.

Although, as I mentioned before I seem to recall (without any refs to
back it up) that there was some work suggesting that luciferin oxidation
in P.stipticus occurred without a dedicated luciferase, rather occurring
as a byproduct of an unrelated pathway. Might be misremembering this one
completely.

[Cathal Garvey]

Naturalism for the win! :D

[Nathan McCorkle]

I've heard fungi using operons....something out of Pam Silver's lab at Harvard

[Dakota Hamill]

Well it looks like they aren't jack-o-lanter's or waxy caps, but  http://en.wikipedia.org/wiki/Mycena_leaiana  They were growing exactly as described, on a deciduous log in a tight clump.  I need to make use of those fungal primers I got..

So...possibly no glow

They did leave an orangey dye stain on my gloves after I was handling them, and from the wiki it says it has mild antibiotic properties, cool stuff. 

[Andreas Sturm]

>An alternative is to perform whole-chromosome digestion/fractionation
>and blunt clone large fragments of DNA into a vector compatible with a
>species more likely to recognise P.stipticus promoters, and then just
>transform and hope for a glowing strain. If you find one, you sequence
>the chromosomal fragment and start guessing which genes encode luciferin
>synthases and luciferases.

That was what I was thinking of.

cut the chromosome in pieces and transform into yeast. Both are fungi, so promotors may work.

If the promotor works, but weakly, then you still can measure light output of the yeast transformant strains. Most will have very little (just background luminescence) and one of the strains may have ten times the background lighting (still far away from being visible). This one will carry the genes / one of the genes for luminescence.


Yielding  cDNA and transforming it into yeast artificial chromosomes (including promoter, RBS and terminator)  could also work. AFAIK, cDNA is very pure (no introns, promoter had not been translated, etc) so you just have the gene itself. But then you have to combine the strains and see which two (or three/ four strains) combined  glow.
 

2012/8/21 Dakota Hamill <dko...@gmail.com>

Well it looks like they aren't jack-o-lanter's or waxy caps, but  http://en.wikipedia.org/wiki/Mycena_leaiana  They were growing exactly as described, on a deciduous log in a tight clump.  I need to make use of those fungal primers I got..

So...possibly no glow

They did leave an orangey dye stain on my gloves after I was handling them, and from the wiki it says it has mild antibiotic properties, cool stuff. 

--

[Cathal Garvey]

In the case of P.stipticus, you've got an odd case as far as mapping
Bioluminescence genes is concerned. The bioluminescent phenotype is
apparently controlled by a *simple* dominant genetic characteristic.
That is, it appears that a single gene, or at least a few closely linked
genes, are responsible for the presence of bioluminescence in
P.stipticus in N.A. samples and the absence in European samples.

This suggests that the gene either:
A) Encodes a protein that forms a normally absent luciferin from
normally present cellular components.
B) Encodes a protein that digests a normally available cellular
components to generate light.

If you can find any work mapping that characteristic to a particular
area of the genome, or even to a specific protein/gene, that's fine. But
it's unlikely to shed any "light" on the other half of the equation.

An alternative in cases like this is to ensure the presence of a known
functional component, and attempt to "knock out" the other unknown half
in a way that makes it easy to detect what knockout caused the
destruction of the phenotype.

One example would be to create a transposon that's compatible with
P.stipticus and contains a copy of the known component of the
bioluminescence system, and use this transposon to randomly integrate
into and inactivate genes in P.stipticus samples.

When you find a non-glowing sample among the survivors (which in itself
is a huge task requiring many, many samples), you perform one of many
"trick" PCRs that allow you to map the location of the transposon in
that case. Such tricks might include digesting the chromosomes with an
enzyme known not to digest the transposon, and re-ligating the fragments
into rings, then performing long-range PCR on the results using primers
for the transposon. This hopefully generates results with transposon
bits at the ends, and flanking regions around the transposon in the
chromosome fused together at a restriction enzyme site.

Using this information, you can then refer back to the P.stipticus
genomic map and locate the gene(s) which, when inactivated, result in a
non-glowing P.stipticus strain, despite the guaranteed presence of the
known dominant characteristic that usually grants a glowing phenotype.

It's messy, and it's time consuming, and if you don't already have a
genomic sequence it's very difficult. It also requires a transposon that
reliably integrates into P.stipticus, though you could probably create
an artificial transposon if you want to shell out the money to have it
synthesised and searched for promiscuous DNA-nicking enzymes.

Given the above, my view is:
If you want a really impressive project (with no guarantee of success)
to map a unique and as-yet poorly understood genetic characteristic to a
gene or set of genes, this might be a lot of fun. It would involve
inventing or refining a set of experimental techniques to work with the
species along the way, and would really legitimise your claim to being a
citizen scientist.

If, however, you want bitchin' glowing organisms on your shelf and you
are looking for a rapid way to achieve that goal, don't go down this
route. It'll burn you out long before you get your glowing pet if you
don't find it interesting. There are 1.5 well understood methods
available off-the-shelf for making things bioluminescent, which have a
huge body of research and documentation to help debug them when things
don't work out. There are off-the-shelf genetic systems for these, and
proven ways of improving light yield, some of which haven't even been
tried in combination yet. Limiting factors in these cases are often
engineering problems you can address to get marked improvements; ways to
culture cells to high densities for longer without acidification of
medium, ways to enhance oxygenation of cells, methods of prolonging
exponential growth or retarding progression to stationary phase, etc.

I *think* you want a bitchin' glowin' thing. In that case, I suggest
buying some P.stipticus spawn for fun, and growing some in your garden
on a waste wood pile, and then continuing to work with the Vibrio
operon. But as I say, if you're looking for an impressively ambitious
science project, you could try mapping P.stipticus.

[Mega]

Guys,
I read by chance in an old thread that the gene for making that un-pronuncable renilla luciferin is known... I am surprised.

As far as I understand, it is the normal GFP with one Serin replaced by Tyr. Just one amino acid????? Do I understand that correct?


We are going to handle agrobacterium soon, so basically I need to get Renilla Luciferase Gene (small chance that it might even be in our lab) and do one point mutation to GFP and this was it? That was the mysterial act of engineering plant bioluminescence??
I could even have printed the sequence out with an 2a-peptide for multicistronic expression, that may be around 2000 nucleotides that beeing 700 euros.



http://www.sumobrain.com/patents/wipo/Bioluminescent-indicator-based-upon-expression/WO1995021191.html

A pre-coelenterazine peptide comprising a modified A. victoria GFP having an amino acid sequence in which Ser65 is replaced with Tyr. There are further provided a polynucleotide encoding the pre-coelenterazine peptide, allowing synthesis of large, pure amounts of coelenterazine, as by culturing organisms transformed with the polynucleotide; methods for synthesizing coelenterazine; and improved assays employing the polynucleotide or transformed organisms, e.g., to detect mutagenesis.

[Mega]

I put that sequence into Genome compiler, Gentle  and online compilers, and all showed the same (so no copy paste problem ;) ):

In the given sequence, there are Stop Codons, and once there is even TAA-ATG, so the ribosome will fall off in eukaryotes, while bacteria may be able to read this next open frame peptide too. (Is there no space needed inbetween?)

It's quite strange because it's not monomeric. Quite difficult to get the sequence I need, but in theory, I'd only need Renilla Luciferase and this Luciferin-making-peptide, put it into a plant nucleus.









On Tuesday, March 15, 2011 10:53:22 PM UTC+1, Cathal wrote:

Brilliant though! That's something to work with at least. There are many things that could have been affecting things to slow down light emission. Codon optimization, protein separation, bottlenecking at any stage.. perhaps by codon optimizing and directly chaining proteins we could get it working? I saw linkage at work on a B12-13 pathway once, it was well over tenfold improvement by my recollection.

Time to read that paper!

On 15 Mar 2011 21:45, "Cory Tobin" <cory....@gmail.com> wrote:

> As to bioluminescent plants, from my understanding they have never yet been
> made to glow indepen...

Sorry to interrupt the conversation on frankenstein experiments. One
small correction about the plants.  There was one case where someone
built the entire luciferin synthesis pathway in plants.
http://dx.plos.org/10.1371/journal.pone.0015461 (PLoS One, open
access)  Unfortunately the amount of luciferin it produced was so low
they could only see the autoluminescence with a five minute exposure
in the dark.  So it wasn't visually stunning :[


-cory

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[Mega]

Guys, there's a new trick for enhancing the bacterial lux operon. 

As you know  there is the luciferase from LuxAB, the decanal from LuxCDE and  you also need FMNH2. 

I found a riboflavin kinase, which converts riboflavin (vitamin b2) into FMN. The fre gene ("Lux G") then reduces the FMN to FMNH2. 

Now the question, should there be additional genes added that produce riboflavin? I think the plant cell will anyways have evolved to produce vitamin b2 if needed urgently? 

[Josiah Zayner]

Plants produce FMN without any exogenous genes I think most every organism does.

[Andreas Sturm]

In the chloroplasts there was much FMN according to the patent of Dr. Krievski.

But in the cell? Well, at least riboflavin (Vitamin B2) sould be there ;)

 

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[Andreas Sturm]

Just came across something I want to share:

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-3040.2002.00889.x/abstract

Maple leaves produce acetaldehyde when transition between light and darkness occurs. That may be used to induce bioluminescence just during the nighttime.

[Nathan McCorkle]

On Sun, Feb 17, 2013 at 4:28 AM, Andreas Sturm <masters...@gmail.com> wrote:
> Just came across something I want to share:
>
> http://onlinelibrary.wiley.com/doi/10.1046/j.1365-3040.2002.00889.x/abstract

http://diyhpl.us/~bryan/papers2/paperbot/2620346323c13aaf83cc73b966566db9.pdf

--
-Nathan

[Dakota Hamill]

I am rooting for you mega!  I want to see some glowy plants from Deutschland!

[Mega]

Thanks ;)

Actually from Austria, but we share the same language (and *some* of the culture) with the Germans :)

[Robert Sidney Cox III]

Nice idea! You could couple this with a circadian promoter....

http://genocon.org/assignment-2012/assignment-a/

[Andreas Sturm]

Well you would have to find a promoter (actually that part would be called an operator, right?)  that switches (the lux genes) on when it "tastes" acetaldehyde. 

Problem, Acetaldehyde may only just be present for <1 hour. So you would have to design a  "self-latching relays" DNA circiuit. But it must get switched of when light comes again, of course... 

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[Andreas Sturm]

A kind of perfect promoter would be this: 

http://partsregistry.org/Part:BBa_K390008

Problem is, it is from cyanobacteria, so it may work in chloroplasts, but surely not in the plant nucleus. 

How it works: When doing photosysthesis, in the cell there are reduced molecules (It comes from Photosystem II IIRC, and I had looked it up that the same photosystem is also present in plant chloroplasts). Those reduced molecules bind to the operator and stop RNA polymerase from working. So as long as photosynthesis occurs, light is off (and some minutes after). 

[Mega]

I just searched for the genome sequence of firefly. Didn't find any results.... Has this never been done? 

What about this: Why not make a genomic library of firefly in Agrobacterium Ti plasmid pGreenII 0049: http://www.pgreen.ac.uk/JIT/pGreenII/T-0049.gif

That would mean, the firefly Luc gene is always present in each colen, and additionally, when the gene for luciferin is present within the restriction fragment, it will make the transformed plant glow then.

Using plants because they are eukaryotic, it may not work in bacteria due to compartimentization. Or would that work better in bacteria, beause everything stays more or less in the cytosol? 

AFAI remember, the 35S Promoter gives *some* expression in bacteria too. But the promoter of the Luciferin gene(s) may not work in bacteria... 

Just some thoughts... 

[Mega]

 *in each clone! ;)

[Nathan McCorkle]

On Sat, Mar 2, 2013 at 2:31 AM, Mega <masters...@gmail.com> wrote:

I just searched for the genome sequence of firefly. Didn't find any results.... Has this never been done? 

What about this: Why not make a genomic library of firefly in Agrobacterium Ti plasmid pGreenII 0049: http://www.pgreen.ac.uk/JIT/pGreenII/T-0049.gif

Screening the library for activity is the hard part, it needs automation because there are millions or billions of clones (library from genomic DNA). I did this by hand one summer in E.coli for some small molecule using an Xgal blue reporter strain... it was not fun.

I really should write some color selective blob counting software....

 

--
-Nathan

[Andreas Sturm]

Screening the colonies for activity in this case would be quite easy. Turn off the lights, and the ones which don't glow are discarded :D

[Patrik D'haeseleer]

That's assuming, of course, that you can get insect genes to express in a bacteria, that they get the correct posttranslational modification to be active, that all the genes for the luciferin biosynthesis pathway are adjacent, and close enough together to fit in the bacteria in one piece, and that the bacteria has the right metabolic precursors available. A *lot* of things would have to go just right for this experiment to work.

At least you do know the firefly luciferase already, so you can add that in on a plasmid.

[Nathan McCorkle]

On Sat, Mar 2, 2013 at 9:02 AM, Andreas Sturm <masters...@gmail.com> wrote:
> Screening the colonies for activity in this case would be quite easy. Turn
> off the lights, and the ones which don't glow are discarded :D
>

Unless you want to calculate transformation efficiency, total
transformants, etc... in that case you need to count ALL bugs, not
just the interesting ones!!!

>
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[Andreas Sturm]

Why would I care for transformation effiecncy? As long as I get plenty of transformants so that each fragment is represented at least once, right? 

that you can get insect genes to express in a bacteria, that they get the correct posttranslational modification to be active, that all the genes for the luciferin biosynthesis pathway are adjacent, and close enough together to fit in the bacteria in one piece, and that the bacteria has the right metabolic precursors available. 

Well, it could be that precursors are already present. Sometimes living organsms use what is available instead of inventing an entirely new metabolic pathway... 

However, maybe, just maybe som leaky expression would be enough... 1 Photon needs just one ATP when using firefly luciferase instead of 60 ATPs for  bacterial luminescence!

So if the attached promoter gives just 1/60 of what the pVIB promoter gives, it would be brightly visible. If it's less, it would still be measurable... 

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[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

> However, maybe, just maybe som leaky expression would be enough...
> 1 Photon needs just one ATP when using firefly luciferase instead
> of 60 ATPs for bacterial luminescence!

Have you got a citation for that? I was under the impression that
bacterial bioluminescence was in the 90-100% efficiency?

> As long as I get plenty of transformants so that each fragment is
> represented at least once, right?

That's what you need high transformation efficiency to guarantee; to
make sure you have clones of an entire genome, you need huge numbers
of transformants.

On 03/03/2013 09:47 AM, Andreas Sturm wrote:
> Why would I care for transformation effiecncy? As long as I get
> plenty of transformants so that each fragment is represented at
> least once, right?
>
> that you can get insect genes to express in a bacteria, that they
> get the correct posttranslational modification to be active, that
> all the genes for the luciferin biosynthesis pathway are adjacent,
> and close enough together to fit in the bacteria in one piece, and
> that the bacteria has the right metabolic precursors available.
>
>
> Well, it could be that precursors are already present. Sometimes
> living organsms use what is available instead of inventing an
> entirely new metabolic pathway...
>
>
> However, maybe, just maybe som leaky expression would be enough...
> 1 Photon needs just one ATP when using firefly luciferase instead
> of 60 ATPs for bacterial luminescence!
>
> So if the attached promoter gives just 1/60 of what the pVIB
> promoter gives, it would be brightly visible. If it's less, it
> would still be measurable...
>
>
>
>
>
>
> On Sun, Mar 3, 2013 at 8:38 AM, Nathan McCorkle <nmz...@gmail.com
> <mailto:nmz...@gmail.com>> wrote:
>
> On Sat, Mar 2, 2013 at 9:02 AM, Andreas Sturm

> <masters...@gmail.com <mailto:masters...@gmail.com>>

> wrote:
>> Screening the colonies for activity in this case would be quite
> easy. Turn
>> off the lights, and the ones which don't glow are discarded :D
>>
>
> Unless you want to calculate transformation efficiency, total
> transformants, etc... in that case you need to count ALL bugs, not
> just the interesting ones!!!
>
>>
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>> the
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>
>
>
> -- -Nathan
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[Andreas Sturm]

> However, maybe, just maybe som leaky expression would be enough...
> 1 Photon needs just one ATP when using firefly luciferase instead
> of 60 ATPs for  bacterial luminescence!

Have you got a citation for that? I was under the impression that
bacterial bioluminescence was in the 90-100% efficiency?

http://mpipz.iwww.mpg.de/25921/koncz_dev_genet_11.pdf

On page "227" (4th page of pdf)

"Comparisons of kinetic parameters and quantum 

yields of luciferases also favoured the firefly enzyme 

which needs only one ATP per emitted photon in con- 

trast to the requirement of 60 ATP for the bacterial 

enzyme "

 

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[Cathal Garvey]

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Wow, I had no idea it was so terrible! How can there be such a vast
gulf in efficiency?

>> <mailto:nmz...@gmail.com <mailto:nmz...@gmail.com>>> wrote:
>>
>> On Sat, Mar 2, 2013 at 9:02 AM, Andreas Sturm
>> <masters...@gmail.com <mailto:masters...@gmail.com>

> <mailto:masters...@gmail.com

> <mailto:masters...@gmail.com>>>
>> wrote:
>>> Screening the colonies for activity in this case would be
>>> quite
>> easy. Turn
>>> off the lights, and the ones which don't glow are discarded :D
>>>
>>
>> Unless you want to calculate transformation efficiency, total
>> transformants, etc... in that case you need to count ALL bugs,
>> not just the interesting ones!!!
>>
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[Andreas Sturm]

True... But as for today, there's no other way to have a full metabolic light pathway...

And obviously it works, just had a flask of glowing bacteria again (revived from the freezer).  It surely drags a lot more energy than neccessary... But imagine how much energy the bacteria could save when switching from lux to luc :D

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[Patrik D'haeseleer]

I think that comparison is completely bogus, since it assumes that you supply the firefly luciferin!

In fact, if you supply the appropriate luciferin for both luciferases, the bacterial luciferase is infinitely more efficient, since the luciferase reaction uses zero ATP, whereas the firefly luciferase reaction uses one ATP.

Just because we know the biosynthetic pathway for the bacterial luciferin, that should not count *against* the bacterial systems' efficiency! For all we know, perhaps it might take 200 ATP to synthesize the firefly luciferin...

Patrik

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[Andreas Sturm]

That's a good point. 

And as long as we don't know the genes responsible for the luciferin, we must take what we have (or supply expensive luciferin!)

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