modified GFP -> pr-coelenterazine peptide

[Mega]

Hi,
does anyone have access to

http://www.sciencedirect.com/science/article/pii/037811199290691H

? (showing the primary structure of wild-type GFP as desrcribed by Prasher et al.)


Searched in the databases our university gives us access to, however no results...
Would be great if someone could help me with that ;)

[Andreas Sturm]

Just found something, finally...

http://www.bio.davidson.edu/courses/molbio/restricted/02gfpseq/gfpseq.html

That should be the wanted paper.

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[Nathan McCorkle]

I found it in HTML via google scholar

http://www.bio.davidson.edu/courses/Molbio/restricted/02GFPseq/GFPseq.html

Here's the crystal structure too:

http://www.tsienlab.ucsd.edu/Publications/Ormo%201996%20Science%20-%20Crystal%20structure.PDF

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[Nathan McCorkle]

I just found the PDF version and it's the same as the HTML one

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[Andreas Sturm]

Thanks for searching!

[Andreas Sturm]

What I really wonder: 

Those jellyfish must produce coelenterazine, obviously, because they're glowing,

So why the wild-type GFP doesn't produce the luciferin? 

I mean, there is just one amino acid (65 Ser -> Tyr) wrong in wild-type gfp, and I consider that highly unlikely that this gene is so very close to producing luciferin, but doesn't. 

Or, does the wild-type GFP produce coelenterazine in small amounts in vivio? Wouldn't this have been measured as GFP is used so frequently? 

Maybe irt's because one usually uses the enhanced GFP, and it does no longer make luciferin?

 

[Andreas Sturm]

Interesting... There is a red-shifted Renilla-Luciferase, which is said to produce a 500-times more light!  Clearly, as the energy stored in red light is smaller there are more photons released. 

http://link.springer.com/content/pdf/10.1007%2Fs11307-011-0515-1

The GFP for producing the coelenterazine would be useless then, though...

[jlund256]

Most/all published protein structures are submitted to the Protein Data Bank:

http://www.rcsb.org/pdb/home/home.do

 

there are tons of GFP structures, see http://www.ncbi.nlm.nih.gov/protein/P42212.1

 

Jim Lund

 

[Cathal Garvey]

GFP really is just green, and fluorescent. However, in A.victoria, it's
complexed to a protein called Aquorin, which is a luciferase, and
produces blue light on digesting its luciferin. This blue light is
partially converted by GFP into green light, producing the turquoise
colour of the jellyfish's glow.

So, GFP on its own neither produces nor consumes a luciferin, but
Aquorin consumes a type of luciferin. However, Aquorin alone does not
produce or regenerate that luciferin, there's a separate pathway for that.

If memory serves, the luciferin for Aquorin is calcium-dependent, so it
is sensitive to membrane ion channels, etc.: I think I remember seeing
Aquorin used as an ionic indicator of some sort.

On 20/12/12 09:26, Andreas Sturm wrote:
> What I really wonder:
>
>
> Those jellyfish must produce coelenterazine, obviously, because they're
> glowing,
>
> So why the wild-type GFP doesn't produce the luciferin?
>
>

> I mean, there is just *one* amino acid (65 Ser -> Tyr) wrong in wild-type

> gfp, and I consider that highly unlikely that this gene is so very close to
> producing luciferin, but doesn't.
>
>
> Or, does the wild-type GFP produce coelenterazine in small amounts in
> vivio? Wouldn't this have been measured as GFP is used so frequently?
> Maybe irt's because one usually uses the enhanced GFP, and it does no
> longer make luciferin?
>

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[Patrik D'haeseleer]

On Friday, December 21, 2012 5:10:20 AM UTC-8, Cathal wrote:

So, GFP on its own neither produces nor consumes a luciferin, but
Aquorin consumes a type of luciferin. However, Aquorin alone does not
produce or regenerate that luciferin, there's a separate pathway for that.

 The luciferin used by A. victoria and Renilla is called coelenterazine, and it kinda looks like three amino acids connected together in a weird way.

It turns out that GFP has to go through some post-translational modifications before it becomes active, including a cyclization reaction in three of the amino acids that make up its chromophore.

The patent that Mega pointed out in the other thread claims that by making just a single amino acid change to GFP, that maturation process will actually produce coelenterazine!

That's why he was asking "So why the wild-type GFP doesn't produce the luciferin?" I suspect that Renilla (and A. victoria) either carry a second modified GFP gene which is used to produce coelenterazine, or they may have some protein-modifying enzyme that can produce the coelenterazine precursor from GFP. They presumably also have enzymes to recycle the oxidized coelenterazine, so you don't have to sacrifice a whole protein for every photon you want to produce.

[Andreas Sturm]

Cathal, are you sure you're not mixing this up?

AFAIK (but I haven't read by far as much as I read about the bacterial lux!)  Coelenterazine can react with renilla luciferase without Aequorin.

In the jellyfish, it is bound to Aequorin and is calcium-dependent.


Yeh, it's strange that it's said that coelenterazine is cut out of modGFP (it doesn't glow anymore then, does it? )  

[Andreas Sturm]

en.wikipedia.org/wiki/Coelenterazine


It is the substrate in many luciferases and photoproteins including Renilla reniformis luciferase (Rluc), Gaussia luciferase (Gluc), aequorin, and obelin.



That seems to be OK...

So basically, if you express modified GFP (= pre-elenterazine peptide) and Renilla Luciferase, that gives you light.

Maybe the modified GFP shifts the light from blue to green?? Or is the new structure not suitable for this? Maybe, too, it's cut out too quick to produce much fluorescence...

[Andreas Sturm]

The jellyfish seems not to make it's own coelenterazine...

The inefficacy of coelenteramide indicates that the medusae are unable to recycle coelenterazine once it is used.

http://www.pnas.org/content/98/20/11148.full

So either you don't need much, or, you they really have to eat much.

[Patrik D'haeseleer]

Interesting - you're digging up some really cool stuff, Mega!

There's a nice parallel here with the (otherwise entirely unrelated) dinoflagellate bioluminescent system. The dino luciferin turns out to be a modified version of chlorophyll, so there's another example where nature figured out a way to reuse an existing photoactive compound as a luciferin. And krill uses a very similar luciferin, which it cannot produce itself (since krill are not photosynthetic) but is thought to aquire through its food - presumably from eating dinoflagellates!

The paper you cited mentioned that "while the ultimate source of the coelenterazine in hydromedusae remains unknown, we believe that crustaceans are the most likely sources", and "the decapod shrimp Systellaspis debilis appears to have the ability to synthesize the molecule". So now the question becomes: can we find a GFP-like protein in these coelenterazine synthesizing shrimp?

[Andreas Sturm]

On Sun, Dec 23, 2012 at 7:00 AM, Patrik D'haeseleer <pat...@gmail.com> wrote:

The paper you cited mentioned that "while the ultimate source of the coelenterazine in hydromedusae remains unknown, we believe that crustaceans are the most likely sources", and "the decapod shrimp Systellaspis debilis appears to have the ability to synthesize the molecule". So now the question becomes: can we find a GFP-like protein in these coelenterazine synthesizing shrimp?

But the question is: Do we need to find that source? Maybe it's quite complicated...

Couldn't one just use the modified GFP to produce coelenterazine? Or a smaller peptide (a fragment of modGFP) which too will make coelenterazine?

[Patrik D'haeseleer]

Speak of the devil - this paper just came out:

Comb Jelly Genome Sheds Light on ... Light

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

They claim this is the first genomic sequence from any bioluminescent animal, and this organism has the same coelenterazine-dependent "photoprotein" luciferases as found in Renilla and A. victoria.

Unfortunately, even though they found *ten* luciferase genes, they did not find any GFP homologs. And they do not know whether this particular species requires coelenterazine in its diet for bioluminescence, or whether it can synthesize the coelenterazine itself - sounds like someone should go figure that out asap. Still, a good first step...

[Andreas Sturm]

How can one beast have ten luciferase genes?



As for the luciferin, the modified GFP may be a shortcut in a metabolic pathway which slowly does the same?


But now that there were no GFP homologues found... strange...

[Patrik D'haeseleer]

Eh, 10 luciferase genes is nothing. In dinoflagellates, the luciferase genes are known to occur in very long tandem repeat regions. Estimates are that P. lunula has at least 200 luciferase genes in a row (Okamoto 2001)! And each luciferase gene itself consists of three copies of the catalytic domain, each of which seems to be catalytically active on its own. It's not known why they have so many copies, but this propensity to gene duplication may be why dinoflagellates tend to have such huge genomes - 60x the size of the human genome!

The comb jelly that was sequenced has a much smaller genome (155 MB, about 1/20th the size of the human genome). The paper does say the luciferases occur in two genomic clusters, comprising three different sequence similarity groups.

Multicellular eukaryotic organisms are quite a bit more complex than bacteria, and it is not unusual to have multiple copies of a gene, each of which may play a slightly different role depending on where and when it is being expressed. In addition, these organisms put up quite an interesting visual display with both bioluminescence and moving light diffraction patterns (if you're ever in California, go check out the jellyfish exhibit in the Monterey Aquarium!). And they also seem to have multiple light receptor genes - the opsins mentioned in the title - so I wouldn't be surprised if they had liciferases that emitted light at slightly different wavelength, and that they used these to communicate.

[Andreas Sturm]

Wow, sounds very complicated. I just had tought of the bacterial Lux-operon, where AB is the luciferase. And everything is in place in one cluster, perfectly compact.

Yeah, I read about retroviruses and retroelements. If there once was a luciferase within a retroelement, that would have given several copies of it - as it is nowadays... 

Maybe, just maybe, this is the way to find the luciferin? Maybe it got also duplicated many times. But very probably it has many genes duplicated which do not play a role in bioluminescence...

[Patrik D'haeseleer]

Chances are the pre-coelenterazine protein will be something that looks a lot like GFP. And if these comb jellies don't have any GFP homologs, I would predict with some degree of confidence that they probably can't make their own coelenterazine, but need to aquire it through their diet. I'm sure someone will be doing that experiment soon, if they haven't already.

[Nathan McCorkle]

Is there possibly a way to search for protein motifs based on the structure of  Coelenterazine or Coelenteramide?

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[Andreas Sturm]

Patrik, that'd be a gret idea!!

We would also see if that p-c-p would give sufficient yield...

[Patrik D'haeseleer]

On Monday, December 24, 2012 11:31:19 PM UTC-8, Nathan McCorkle wrote:

Is there possibly a way to search for protein motifs based on the structure of  Coelenterazine or Coelenteramide?

Not much of one. The chromophore core in GFP is Serine-Tyrosine-Glycine, which becomes Tyrosine-Tyrosine-Glycine in the patent Mega dug up. You also need a phenylalanine in position 64 that becomes part of the coelenterazine. So the core motif you're looking for is FYYG, but that doesn't narrow it down by much.

Presumably you need the rest of the structure of the GFP to make the cyclization reaction possible, so you're better off looking for a GFP homolog, with FYYG where you'd expect to see FSYG.

[Andreas Sturm]

Presumably you need the rest of the structure of the GFP to make the cyclization reaction possible,

In the patent there is also a claim that the patent a peptide from 64 to 69, so *perhaps* you really would just need this oligopeptide?


But there is a very big question:
The pre-czn-peptide is based on this
http://www.bio.davidson.edu/courses/molbio/restricted/02gfpseq/GFPseqfig2.GIF

Nucleotide 736-739 (in frame!) is TAA so the jellyfish polymerase will stop and fall of the mRNA. Then it goes on with ATG TCC ..... That is junk DNA? I would expect it to be an apo-protein or something alike. But jellyfish machinery doesn't support operons, so this wouldn't be expresssed.

Or is this a compilation, and before the ATG there was another promoter?

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[Andreas Sturm]

Presumably you need the rest of the structure of the GFP to make the cyclization reaction possible,

In the patent there is also a claim that they patent a peptide from 64 to 69, so *perhaps* you really would just need this oligopeptide?

But there is a very big question:
The pre-czn-peptide is based on this
http://www.bio.davidson.edu/courses/molbio/restricted/02gfpseq/GFPseqfig2.GIF

Nucleotide 740-743 (in frame!) is TAA so the jellyfish polymerase will stop and fall of the mRNA. Then it goes on with ATG TCC ..... That is junk DNA? I would expect it to be an apo-protein or something alike. But jellyfish machinery doesn't support operons, so this wouldn't be expresssed.

Or is this a compilation, and before the ATG there was another promoter?

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[Andreas Sturm]

If you were to synthesize the sequence, would you trust this source - for the luciferase:

http://www.stanford.edu/~loening/plasmids/sequence/RLuc8.6-535.fasta

>RLuc8.6-535

 

MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNATSSYLWRHVVPHIEPV ARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGSALAFHYAYEHQDRIKAI VHMESVVDVIESWMGWPDIEEELALIKSEEGEKMVLENNFFVETLLPSKIMRKLEPEEFAAYLEPFKEKG EVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVK VKGLHFLQEDAPDEMGKYIKSFVERVLKNEQ

( from http://www.stanford.edu/~loening/plasmids/index.html )


??

Best,

[Nathan McCorkle]

Hrmm, I can't seem to find the patent mention.... I'm wondering if that cyclization is spontaneous or not... If not I'd want to search for proteins with a binding pocket shaped like the phenol and phenyl side chains in coelenteramide and coelenteramine (the side away from the amide/amine in the metabolites).

If the cyclization is spontaneous, maybe you could fuse the luciferase to an ubiquitinization enzyme, to immediately mark the luciferin-protein for recycling once it was used for photon emission

.

On Wed, Dec 26, 2012 at 9:21 AM, Andreas Sturm <masters...@gmail.com> wrote:

ptide from 64 to 69, so *perha

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[Patrik D'haeseleer]

On Wednesday, December 26, 2012 9:21:51 AM UTC-8, Mega wrote:

But there is a very big question:
The pre-czn-peptide is based on this
http://www.bio.davidson.edu/courses/molbio/restricted/02gfpseq/GFPseqfig2.GIF

Nucleotide 736-739 (in frame!) is TAA so the jellyfish polymerase will stop and fall of the mRNA. Then it goes on with ATG TCC ..... That is junk DNA? I would expect it to be an apo-protein or something alike. But jellyfish machinery doesn't support operons, so this wouldn't be expresssed.

No time to look it up (my wife says "no work on vacation!" and this is starting to look too much like work ;-), but make sure you're using the right genetic code - jellyfish may well use something nonstandard - and take any introns into account.

The GFP sequences we use every day have typically been recoded for E. coli, so they won't have any nonstandard codons or introns.

Patrik

[Jordan Miller]

FYI: our extremely capable iGEM team tried the mod GFP to make pre-coelenterazine a while back and the patent appeared not to be true in our hands (it was not usable as a substrate for Gaussia or Renilla luciferase, we used a luminometer and all the proper controls). We had asked the inventors and they told us IIRC that they had not pursued it nor proven it since then. So it appears to be a strictly theoretical patent rather than a real coelenterazine synthesis pathway and in our hands it was not a worthwhile venture. Still no papers based on this patent of mod-GFP either.

soooo... good luck!!

jordan

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[Patrik D'haeseleer]

Heh - good to know! Yeah, I was wondering why there were no papers on this whatsoever. Because if this worked, that would be big news. And with the patent out, they'd have no reason *not* to publish it...

Patrik

[Andreas Sturm]

But why did they patent it then? 

Jordan, it would be interesting how you did that... Did you take wild-type GFP and make a point mutation? Or enhaced GFP?

Have you used the very strongest promoter (maybe the luminescence was too weak to be mesaured?) ?

P.S. "Sciencenewstop" is not part of google, is it? I just found this... They didn't even care about deleting email adresses cited...

 http://sciencenewstop.blogspot.co.at/2012/12/re-diybio-modified-gfp-pr_705.html 

[Jordan Miller]

we did the point mutation on eGFP optimized for bacterial expression with the induced vs. non-induced controls and coelenterazine as the positive control with purified Gaussia luciferase from a mammalian expression system. did not work as expected in our hands.

the ability to get this would revolutionize non invasive imaging since you wouldn't need to inject any luciferin/coelenterazine, so of it did work it would have been a big paper by the inventors. the most worrisome thing to me is they appear to have dropped the project.

jordan

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[Andreas Sturm]

On Thu, Dec 27, 2012 at 7:49 PM, Jordan Miller <jrd...@gmail.com> wrote:

we did the point mutation on eGFP optimized for bacterial expression with the induced vs. non-induced controls and coelenterazine as the positive control with purified Gaussia luciferase from a mammalian expression system. did not work as expected in our hands.

Ok, but the sequence of eGFP differs from natural wild-type GFP... You just changed Amino acid 65 Ser -> Tyr ?

the ability to get this would revolutionize non invasive imaging since you wouldn't need to inject any luciferin/coelenterazine, so of it did work it would have been a big paper by the inventors. the most worrisome thing to me is they appear to have dropped the project.

This, however, is a very good point. It truely would be a revolution.

But I had had a research some times ago on how much a patent costs, and it's horrible. And, there's no world-wide patent,  you have to buy it for each country seperately. So you hardly get a patent in most of the countries (US, some European countries, Brazil, Russia) below 10'000 $ !

Why would they throw away > 10k $ for patenting something that doesn't work??



Did you get really *no* luminescence? Had a HPLC on it?

[Nathan McCorkle]

Seems that this whole patent-not-working points to the coelenterazine not spontaneously forming from the modGFP alone. I bet there are some other enzymes in there that recycle/recharge the modGFP

--

[Jordan Miller]

it doesn't make evolutionary sense though. GFP was optimized during evolution for fluorescence not coelenterazine. do other coelenterates have GFP-like genes?

and in vivo coelenterazine synthesis has not been figured out AFAIK.

the patent is a hypothesis, not a proof of concept or reduction to practice.

jordan

[Andreas Sturm]

But if the patent is just a hypothesis:

I understood while reading the patent, that on the end there was written that they already did it. At least, it sounded to me as if they would have tried it already with success. Have to read it again.

[Andreas Sturm]

The luminometer gives a reading of 1.5×10⁷ hv/sec upon addition of luciferase, against a background of 4×10⁵ hv/sec. The read-out from this instrument appears in FIG. 3.

That looks very much as it would have worked?

Or they were were lying - but why would they?

[Dakota]

Apologies ahead of time if this is not related as I havn't caught up fully on this thread.

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

they managed to do point mutations with gibson assembly I think, and get colors changes.  

This might also be of some interest, but the links within it seem to be N/A  http://dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.cryst.bbk.ac.uk/projects/gmocz/gfp.htm

[Andreas Sturm]

Yeah, that's for the luciferase, but colour of the light will be our smallest problem...
(One should insert such modified firefly luciferases into fireflies to get bugs glowing in different colours :D )

[Nathan McCorkle]

Does the patent say the modGFP was extracted from wildtype organism or overexpressed in a lab workhorse strain?

[Andreas Sturm]

They used wild-type A.Victoria GFP, and exchanged 65 Serin to Try.

The patent mentions using promoters other than native GFP promoter

e.g. here

3. The method of claim 2 wherein means for effecting expression of said polynucleotide is E. coli strain BL21 (DE3)Lys S transformed with an expression vector comprising, 5' of said one or more sequences of nucleotide bases collectively encoding the amino acid sequence of pre-coelenterazine peptide, one or more appropriate regulatory control sequences which collectively enable expression of said polynucleotide; and one or more sequences of bases which collectively confer resistance to an antibiotic upon an organism. 
4. The method of claim 3 wherein said means for effecting expression of said polynucleotide is the E. coli SMC2 (ATCC Accession No. 69553). 

It seems they used the cDNA 

To generate a polynucleotide having these encoding sequences, one may introduce one or more point mutations into the cDNA for the GFP of A. victoria in the cDNA described in Prasher, et al., supra, using in vitro mutagenesis methods well known to those skilled in the art.

Btw., 

Parent Case Data: This application is a division of application Ser. No. 08/192,158, filed Feb. 4, 1994 now abandoned.

Patent invalid?  

Oh god, 

 This code is degenerate; thus, the Arg at residue 109 may be encoded by nucleotide bases as CGT, but could, under the code, equally be CGC, CGA, CGG, AGA or AGG, and still encode for Arg. Similarly, nucleotide bases encoding Tyr as TAT could equally be TAC and still encode Tyr for residue 65 in the peptide. Alternatively, the polynucleotide may be a cDNA (or RNA equivalent) which includes, in addition to the nucleotides for Ser⁶⁵ being altered from TCT to TAT, mu

It feels like 90% of the patent explained something obvious for people who are a bit familiar with biology....  

[Andreas Sturm]

Honestly, this 

Suitably, the regulatory element may be a promoter. Suitable promoter elements include a promoter activated by heavy metal (e.g. the one described in Freedman, et el. J. Biological Chemistry, 268: 2554, 1993, incorporated herein by reference); a P 450 promoter (e.g. the cytochrome P 450); or a promoter for a stress protein, (e.g., described in Stringham, et. el., Molecular Biology of the Cell, 3: 221, 1992), one of said stress proteins being a heat-shock protein. Other suitable promoters include that of the arabinose operon (phi 80 dara) or the colicin E1, galactose, alkaline phosphatase or tryptophan operons. Similarly the ADH system may be employed to provide expression in yeast. Alternatively, the regulatory element may be an enhancer.
The regulatory control sequences are operatively linked with the polypeptide comprising one or more sequences of nucleotide bases collectively encoding an amino acid sequence of a pre-coelenterazine peptide; i.e., the regulatory control sequences are placed on the polynucleotide at a distance 5' of the one or more sequences suitable to enable expression of the sequences.
Polynucleotides which bear one or more of such regulatory control sequences may be used in transforming organisms, as when suitably the polynucleotide is included in an expression vector. The regulatory control sequences are selected for compatibility with the organism into which the polynucleotide is to be incorporated by transformation, i.e., the regulatory control sequences are those which may be recognized by the transformed organism or cell and which will aid in controlling the expression of said polynucleotide in the transformed organism.
Thus when the organism to be transformed is E. coli, the regulatory control sequence may be a promoter (e.g., the T7, the SP6 or lac promoter); or transcription initiation sequences for ribosome binding (e.g. the Shine-Delgarno sequence and the start codon AUG). When the organism to be transformed is eucaryotic, the regulatory control sequences may include a heterologous or homologous promoter for RNA polymerase II and/or a start codon AUG. For example, when the target of transformation is a mammalian cell, the regulatory control sequence may be a promoter (e.g. the SP 40 or the bovine papilloma virus promoter). Suitable regulatory control sequences for use in other microbe, or in animal, or plant cells may be selected according to criteria well known to persons having skill in the art. All of these regulatory control sequences may be obtained commercially (individually or incorporated into a vector) or assembled by methods well known in the art.
Where the polynucleotide carries one or more appropriate regulatory control sequences, there may further be present one or more further sequences of bases which collectively confer resistance to an antibiotic when the polynucleotide is expressed in an organism. Such genes for antibiotic resistance are desirable components for expression vectors since they facilitate identification of transformed cells grown in the presence of antibiotics, and exert a continual pressure on the transformed organisms to retain and express the expression vectors.

could have been left out. It should have been left out, to make it understandable, 

[Patrik D'haeseleer]

Remember, you're not reading a scientific paper, you're reading a *patent*. Patents are not written to be legible, but to be legally defensible. Patent lawyers play by entirely different rules, which entirely too often seem completely nonsensical.

[Andreas Sturm]

Jordan:

 

we did the point mutation on eGFP optimized for bacterial expression with the induced vs. non-induced controls and coelenterazine as the positive control with purified Gaussia luciferase from a mammalian expression system. did not work as expected in our hands.

But, are you *sure* that with wild-type GFP there would also not be luciferin production? 

It's worth too much to not trying it.... It would be the first (and only) eukaryotic light generating pathway, consisting of only two (!) genes... Too good to be true??? 

[Jordan Miller]

no I'm not sure. but yes it does seem too good to be true.

if you get it to work let us know!

luminometer has the highest signal to noise of any assay so I'm positive it didn't work how we did it.

jordan

--

[Andreas Sturm]

I just got a ad-email from GeneArt telling me that  in my country there's special condition for academic labs... only 25 cents per base pair but limited in time. If I knew it would work, I'd have it synthesized immediately with the super-strong plant promoter and optimized renilla luciferase.

(In total that would be 625€ which I'd consider acceptable prize for such a project)


But - as said, if there's low (or no) chance that it works, it'd be pointless waste of rare money.

[Eugen Leitl]

On Sat, Dec 29, 2012 at 12:19:58PM +0100, Andreas Sturm wrote:
> Honestly, this

...

>
>
> could have been left out. It should have been left out, to make it
> understandable,

The only time you learn is when you're pushed out of your
comfort zone.