Phosphorescent Proteins and Plants

[Sebastian S. Cocioba]

I was thinking about this for a while now. What about forgetting auto-luminescent plants and trying phosphorescent plants instead? I spoke with some colleagues and they all said to keep it hush hush in case it works and patent the crap out of it. I would rather have the brilliant minds of the DIYBio community take a crack at the idea and if it works then go open source. Anywho, my question is: Does anyone know of phosphorescent proteins that could be expressed in plants? Charge during the day, glow at night. Ive seen some papers on non-exponential light release with phosphorescent molecules but they were all kinds of salts. Ive also heard that high amounts of tryptophan residues have a visible phosphorescent activity. The pipe-dream of high light output via lux pathway seems to be just that. As another DIYBiologist said, the metabolic rate required for sustained illumination would kill the plant...unless there is something we are overlooking of course.

Sebastian S Cocioba
CEO & Founder
New York Botanics, LLC

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[Cathal Garvey]

Neat idea! Anyone out there understand this stuff enough to contribute?

It's possible that you could do light "storage" in crippled fluorescent
molecules, for example antennae proteins from algae without
corresponding chlorophyll to "dump" excited electrons on; you might be
able to hack them to deliver that energy to a fluorophore over time via
quantum tunneling or what-have-you; tweak the availability of said
fluorophore to get the light-emission rate you want.

Chief challenge in that case would be trying to engineer or evolve
antennae proteins that stably retain their excited state without
decaying or dumping the electron on inappropriate molecules. I'm no
chemist, but maybe you could "convince" your antennae to store the
electrons in aromatic carbon rings in an otherwise aromatic-ring-free
protein core until a fluorophore binds the target site and comes within
tunneling range?

This is me probably talking complete nonsense. There's nothing more
dangerous than someone who knows the jargon without understanding any of
the concepts. :P

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[Patrik D'haeseleer]

I would guess that light given off by phosphorescent proteins will be much weaker than fluorescence, since the light is emitted over a much longer time period. That's just mu guess though...

On Saturday, October 20, 2012 10:20:15 AM UTC-7, Sebastian wrote:

The pipe-dream of high light output via lux pathway seems to be just that. As another DIYBiologist said, the metabolic rate required for sustained illumination would kill the plant...unless there is something we are overlooking of course.

This part I completely disagree with. As I mentioned before, the Cambridge iGEM10 "E. glowli" team showed that the energy required for bioluminescence in plants should definitely be feasible. And if you aim to just have small patches that light up for an hour or two, you should be able to get away with orders of magnitude less energy.

Just because nobody has done it doesn't mean it's impossible! I expect we'll need to do at least a little metabolic optimization, and careful targeting of plant organs or cell types using appropriate promoter sequences to get a strong light output. But I wouldn't be surprised if you could get visible results even with a fairly naive approach.

Patrik

[Sebastian S. Cocioba]

http://sphotos-a.xx.fbcdn.net/hphotos-snc6/s480x480/184113_508669685828140_140086363_n.jpg

Driveway stones. Phosphorescent rocks. Fluorescent pigments need UV. Bioluminescence causes emission via an ongoing chemical reaction. Where would you classify luciferin oxidation? It does not store light, and it does not use light to excite and emit other light so Idk but if there are some kind of protein based phosphorescent molecules that we could isolate and express with a simple chloroplast gene expression pathway, it might work. I gotta hit da books and see but it would be nice to make it happen. I bet a few hotels would love some on their premises. Ill post if I find anything regarding this. 

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[Patrik D'haeseleer]

Yeah, phosphorescence in various chemicals works quite spectacularly. But you have to keep in mind that you have store all that energy in the molecule in some way. And keeping some molecule in a charged state for a long time may not be that compatible with biology.

Of course, the alternative to storing the energy in a single molecule, would be to store the energy using some chemical reaction. But that is exactly what bioluminescence does. In fact, the dinoflagellates we're studying use chlorophyll to store solar energy during the day, producing oxygen along the way, and they actually use a modified version of their chlorophyll as a luciferin at night, using oxygen to create light:

[Mega]

The usual fluorescent pigment used is Strontium-aluminate. That'd be quite hard to prodce for plants.

If there is a protein which already does this, that'd be great. But I fear there just hasen't been need for it in nature.
You'd have to design your own protein which will be phosphorescent... Computer simulations are still far off from this scenario, it's a pity.

[Rikke Rasmussen]

How well described is the bioluminecensce in dinos? Could it be transformed into plants?


/Rikke

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[Patrik D'haeseleer]

On Sunday, October 21, 2012 7:56:21 PM UTC-7, Rikke wrote:

How well described is the bioluminecensce in dinos? Could it be transformed into plants?

Not yet. Algae have huge genomes and this one hasn't been sequenced yet. We know the luciferase gene and the structure of the luciferin, but we don't know the genes that turn chlorophyll into luciferin and back. We're hoping to look for some of the other genes involved at BioCurious, but frankly it's a bit of a fishing expedition, and likely to be technically challenging.

I think it basically boils down to trying to do some transcriptomics (EST sequencing?) or proteomics (2D-DIGE, or a pulldown using purified chlorophyll or luciferin). Either of which would probably be a first for a DIY lab.

Anyone have any suggestions for other approaches to identify the luciferin biosynthesis pathway?

[Andreas Sturm]

That's a great idea!! As an algae it has the cellular structure and mechanism of higher plants! The genes are likely to work quite 1:1.

But: Why do we know it's not their chloroplasts which glow? 

(Inserting their chloroplasts into a plant cell sounds doable. There once was a paper about plastidal chimeras - exchanging chloroplasts - in plants which may occur when grafting plants.)

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[Cathal Garvey]

You could save even more energy plant-wise if you made them
touch-sensitive, so they only glow for a while when brushed against.
Non-trivial, obviously, but entirely doable; look at the "sensitive
plant" and venus flytraps, not to mention the threadlike grippers of
many climbers, for examples of ubiquitous touch-sensing in plants.

You could also steal those poorly-understood sound-detecting systems
from wheat etc. and plug them in, so that you'd have an ultrasound
"light switch" to activate bioluminescence. :P

Although, not so happy for the environment, blaring out ultrasound like
that. Noise pollution for rodents, bats and birds.

On 21/10/12 00:06, Patrik D'haeseleer wrote:
> I would guess that light given off by phosphorescent proteins will be much
> weaker than fluorescence, since the light is emitted over a much longer
> time period. That's just mu guess though...
>
> On Saturday, October 20, 2012 10:20:15 AM UTC-7, Sebastian wrote:
>>
>> The pipe-dream of high light output via lux pathway seems to be just that.
>> As another DIYBiologist said, the metabolic rate required for sustained
>> illumination would kill the plant...unless there is something we are
>> overlooking of course.
>
>
> This part I completely disagree with. As I mentioned before, the Cambridge

> iGEM10 "E. glowli" team <http://2010.igem.org/Team:Cambridge/Tools/Lighting>showed that the energy required for bioluminescence in plants should

> definitely be feasible. And if you aim to just have small patches that
> light up for an hour or two, you should be able to get away with orders of
> magnitude less energy.
>
> Just because nobody has done it doesn't mean it's impossible! I expect
> we'll need to do at least a little metabolic optimization, and careful
> targeting of plant organs or cell types using appropriate promoter
> sequences to get a strong light output. But I wouldn't be surprised if you
> could get visible results even with a fairly naive approach.
>
> Patrik
>

[Andreas Sturm]

Plants saving energy will be the generation 2.0 ;) 

[Andreas Sturm]

But actually, a great idea!

[Rikke Rasmussen]

Depending on how complex the pathway is, a pull-down assay using chlorophyll might work (I'm assuming you know for sure that chlorophyll is indeed a precursor to luciferin) - it might at least give you the first enzymatic step. You could probably save yourselves considerable amounts of time by doing some bioinformatics homework first, looking for proteins in other algae/plant species that bind chlorophyll, then go back to the encoding sequences (or make a qualified guess at the mRNA sequences) to create PCR primers and use those to look for analogs? Then clone, purify, add chlorophyll, and analyze with NMR/HPLC/whatever (not a chemist, here) to get some clue of what chlorophyll is initially converted into.

Seeing as my imagination is now solidly captured by the topic, do you happen to have any good papers on luciferin in dinos? Also, which species are you working with? A quick-n-dirty Google scholar search revealed that molecular markers have been developed for at least some species, and may come in handy.

And if anyone can get their hands on this paper, it promises to be a review of microorganism biosynthesis of marine natural products - it may give further clues to the luciferin pathway in dinos.

/Rikke
 

On Mon, Oct 22, 2012 at 7:33 AM, Andreas Sturm <masters...@gmail.com> wrote:

But actually, a great idea!

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[Conner Berthold]

"The complete primary sequence of the 238 amino acids of Aequorea green fluorescent protein (accession P42212) was not revealed until the cloning and sequencing of its cDNA by Prasher in 1992 (Prasher et al. 1992). Just two years later came the first dramatic demonstrations that the gene was self-sufficient to undergo the post-translational modifications necessary for chromophore formation. Specifically, Chalfie reported the gene encoding Aequorea green fluorescent protein could be functionally expressed in the sensory neurons of the worm Caenorhabditis elegans (Chalfie et al. 1994) and Inouye and Tsuji showed that expression of the gene in Escherichia coli resulted in green fluorescent bacteria (Inouye and Tsuji 1994)." (link)

and...

"changes in membrane permeability of the cells in the pulvini occur that allow for the rapid movement of calcium ions.  This has been related to increased cell wall pliability in the pulvini, which when coupled with decreased turgor pressure allows for movement." (link2)

The main trigger for the reaction being the addition of Ca2+ as a promoter. Sensitive plant also uses Calcium ions as their trigger, so if you could make it activate the fluorescent protein instead of (or both?!?)a  kinetic movement the plant would "light-up" when touched/agitated.

[Sebastian S. Cocioba]

Im working on isolating pulvini cells from mimosa pudica at the moment. They are easily recognized by their reddish pigments and cluster in small groups near the area where the leaf petiole meets the stem. An idea would be for me to hold off on the glow aspect and try to casette the quake sensing ion channels. Touch based glow would be wonderful. Imagine a weeping willow that bursts with a soft pink light when a breeze blows its branches. :) from what I understand, the actual mechanoreceptor has yet to be isolated so that is a potential field of work. Also the signal transduction starts at any point and then the pulvini collapse. They signal can also be triggered with electric shock and fire. The response time of the leaves depend on net light exposure so low photoperiod plants would need quite the stim to respond ad will do so slowly. Cool stuffs!

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[Patrik D'haeseleer]

On Monday, October 22, 2012 3:36:16 PM UTC-7, Rikke wrote:

Depending on how complex the pathway is, a pull-down assay using chlorophyll might work (I'm assuming you know for sure that chlorophyll is indeed a precursor to luciferin) - it might at least give you the first enzymatic step.

 I believe the gene for one of the initial steps is actually known, but there's still several steps after that to get to luciferin.

You could probably save yourselves considerable amounts of time by doing some bioinformatics homework first, looking for proteins in other algae/plant species that bind chlorophyll, then go back to the encoding sequences (or make a qualified guess at the mRNA sequences) to create PCR primers and use those to look for analogs?

Seems unlikely to be successful, since there are no closely related sequenced strains, and we don't necessarily know the chlorophyll binding proteins in those strains either.

Seeing as my imagination is now solidly captured by the topic, do you happen to have any good papers on luciferin in dinos? Also, which species are you working with? A quick-n-dirty Google scholar search revealed that molecular markers have been developed for at least some species, and may come in handy.

I sent you an invite for the Mendeley group we've put together with a bunch of references. It's a private group, so we can share pdfs, but that also means there's a limited number of people who can join.

If anyone else would like to be added to our Bioluminescence Mendeley group, let me know...

We're working mostly with Pyrocystis lunula. Pyrocystis seems to be the only cultured genus that is nontoxic, it's readily available from http://empco.org , and there is a set of circadian EST sequences for P. lunula that may prove useful for identifying some luciferin biosynthesis genes as well. 

[Rikke Rasmussen]

Too bad doing large-scale mutant screenings on single-cell aquatic organisms is likely to be the royal pain of a life-time - that would've made life so much easier. If anyone can come up with a good way to do this, though, pipe up.

Patrik, thumbs up for Mendeley, that looks sweet! I'll start reading up, see if I might be able to add some useful brain power to this project. 

Since the Mimic Mars project seems to have suffered a small set-back in enthusiasm due to the high hardware entry barrier, could the elucidation of the biosynthetic pathway of luciferin in P. lunula (and perhaps/probably other related dinoflagellate species) come into consideration as the international DIYbio project? Perhaps with the added perk that that knowledge would then perhaps allow us to create the bioluminescent plants we keep on talking about (because yes, I totally want those, too - goodbye streetlights and night time park stalkers...no more hiding in the bushes...ever!).

We could set out to full genome sequence, develop molecular markers, create BAC/YAC libraries and lots of other goodness, depending on skill and resources - there'd also be lots of work for those with more inclination for software than wetware in the data analysis, annotation and bioinformatics.

For a single academic lab, this would take years...but aren't we out to prove the efficacy of distributed effort among enthusiastic citizen scientists?

/Rikke

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[Patrik D'haeseleer]

I don't think P. lunula genetics would make for a great international DIYbio project. It's at least an order of magnitude harder than, say, characterizing genes in Arabidopsis, where the whole genome is known and there's all sort of genetic tools already available.

*Growing* P. lunula is something far more accessible though. And there's some nice experiments that we're planning to start doing at BioCurious, including testing cocultures with marine bacteria, measuring how fast the dinos regenerate their luciferin after being shaken, how fast they turn off their bioluminescence after being exposed to light at night, what light frequencies they respond to, etc.

Patrik

[Andreas Sturm]

I don't think P. lunula genetics would make for a great international DIYbio project. It's at least an order of magnitude harder than, say, characterizing genes in Arabidopsis, where the whole genome is known and there's all sort of genetic tools already available.

Well, surely it is kind of the advantage DIYBio has, that it builds up on existing things such as pre-sequenced organisms.

Standing on the shoulders of giants, as we say.


But, however, it seems to be complex. IIRC: They don't just produce the substances and put them into the cytoplasma like vibrio fischeri.
They make inclusion bodies that glow. But maybe I remember wrong.

[Rikke Rasmussen]

Fair enough, that's a valid point. Not exactly unaware of the complexity level, as I'm old enough to remember the days before full-genome sequencing and the challenges that involved. But there is no doubt that it would be both long-term and difficult for DIYbio to do  something like this all on our lonesome - it'd definitely require fairly liberal access to bigger, better-equipped labs, and I guess if we had that on hand, most of us wouldn't be tinkering in the garage.

/Rikke
 

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[Cathal Garvey]

Nice observation! So use a Ca-sensitive luciferase and you might get transitory glowing after contact as-is. Not worth the money just to risk it, but as costs of synthesis fall it'll eventually be worth it just to see if it works!

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[Andreas Sturm]

Yeah, one day when it costs just 100€, it's definitely worth it ;)

Is that day so far away?

[Mega]

Here:

Scintillons  are that inclusion  bodies that glow. Daytime, chloroplasts are at the cell membrane, nighttime, they wander to the nucleus. Scintillons do the other way round. 

http://yubanet.com/scitech/Suggested-Explanation-for-Glowing-Seas----Including-Currently-Glowing-California-Sea_printer.php

http://www.ncbi.nlm.nih.gov/pubmed/8398076

[Mega]

I wonder if you could take dinoflagellate chromosomes and put it into plant nucleus?

That would give you polyploid plants that glow when touched??

[Sebastian S. Cocioba]

The mechanism for touch has not yet been fully isolated and introduced into another organism. I believe dinos act like other marine bioglowers and have a quorum sensing based glow response so touch would not work since it is not there. I could be wrong. If one could isolate the theoretical membrane bound mechanoreceptors from touch plants and tie it in with a glow promoter in the chloroplast, that might work. Maybe take animal mechanoreceptors and tie them to hair promoting genes like those from tomato or african violet. Then do the whole glow response jazz. Many possibilities. Sty for the tangental response, mega. :)

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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On Nov 2, 2012, at 4:31 PM, Mega <masters...@gmail.com> wrote:

I wonder if you could take dinoflagellate chromosomes and put it into plant nucleus?

That would give you polyploid plants that glow when touched??

On Saturday, October 20, 2012 7:20:15 PM UTC+2, Sebastian wrote:

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[Patrik D'haeseleer]

On Friday, November 2, 2012 1:54:05 PM UTC-7, Sebastian wrote:

The mechanism for touch has not yet been fully isolated and introduced into another organism. I believe dinos act like other marine bioglowers and have a quorum sensing based glow response so touch would not work since it is not there.

Nope. The dinoflagellate bioluminescence is indeed triggered by mechanoreception. You're thinking of bacterial bioluminescence in Vibrio, which is indeed triggered by quorum sensing. Completely independently evolved bioluminescence systems.

Sensing the mechanical stress happens in the cell wall, and since plants have a *very* different cell wall, there is no reason to believe that randomly inserting dino chromosomes into plant cells would do anything but completely screw up the cell and kill it.

It's a bit like taking a jet engine, cutting it into pieces, shoving a chunk underneath your car's hood, and expecting it to fly...




 

[Michael Fridman]

"It's a bit like taking a jet engine, cutting it into pieces, shoving a chunk underneath your car's hood, and expecting it to fly..."

I like that analogy. I've been tweaking with dumping some bacterial genes into plants and hoping for bioluminescence. But your analogy summarizes the challenges well. The whole system needs to be "built" from the ground up for that particular purpose. Anyways, not sure if this is the right topic for this discussion, as the main question was whether plants can utilize chemiluminescent molecules, such as strontium aluminate or zinc sulfide...