RE: [DIYbio] Re: Small side project - baking GFP bread - exhibtion
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Sebastian Cocioba
Dec 9, 2013, 9:32:20 PM12/9/13
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Just remember that some GFPs are sensitive to pH and will denature in acidic conditions. Maybe even the high temp of baking? Either way it would be neat to see if you can get decent expression. Best of luck!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Mega [Andreas Stuermer]
Sent: 12/9/2013 2:11 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Re: Small side project - baking GFP bread - exhibtion
As the fluorescent bread is approaching (hope to get it done before Christmas), I am thinking how I will breed the bugs.
I will innoculate about one liter of yeast medium from a petri dish.
In our lab exercise, we had professional fermenters. At home it would be possible to build one, as I have two air pumps lying around in the basement - just attach a sterile filter.
But I think the cheapest and quickest idea is to keep them in very big containers, with the liquid being just around two centimeters high. Thus free diffusion would be sufficient.
But regulating the pH value without contaminating it will be another issue. Wouldn't it be better to skip pH regulations? Add some Penicillin-G (it should decay during baking) and 8% ethanol. The ethanol will kill most of the bacteria anyway.
The initial pH would be around 5.5, so the yeast has an initial propagation advantage over many bacteria. The ethanol will do the rest.
After some time, the ethanol will be consumed aerobically by the yeast. But it will outgrow any bacteria before they get a chance to propagate I assume.
-- I will innoculate about one liter of yeast medium from a petri dish.
In our lab exercise, we had professional fermenters. At home it would be possible to build one, as I have two air pumps lying around in the basement - just attach a sterile filter.
But I think the cheapest and quickest idea is to keep them in very big containers, with the liquid being just around two centimeters high. Thus free diffusion would be sufficient.
But regulating the pH value without contaminating it will be another issue. Wouldn't it be better to skip pH regulations? Add some Penicillin-G (it should decay during baking) and 8% ethanol. The ethanol will kill most of the bacteria anyway.
The initial pH would be around 5.5, so the yeast has an initial propagation advantage over many bacteria. The ethanol will do the rest.
After some time, the ethanol will be consumed aerobically by the yeast. But it will outgrow any bacteria before they get a chance to propagate I assume.
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Cathal Garvey (Phone)
Dec 10, 2013, 4:06:36 AM12/10/13
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IIRC once folded, GFP is somewhat stable up to 60C: could try a weird baking cycle of rise->cook to heat innards for long time at 40 then 50, then grill the outside to form crust?
I'm not convinced you'll get edible results even if it were legal to try it!
I'm not convinced you'll get edible results even if it were legal to try it!
Sebastian Cocioba <scoc...@gmail.com> wrote:
Just remember that some GFPs are sensitive to pH and will denature in acidic conditions. Maybe even the high temp of baking? Either way it would be neat to see if you can get decent expression. Best of luck!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Mega [Andreas Stuermer]
Sent: 12/9/2013 2:11 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Re: Small side project - baking GFP bread - exhibtion
As the fluorescent bread is approaching (hope to get it done before Christmas), I am thinking how I will breed the bugs.
I will innoculate about one liter of yeast medium from a petri dish.
In our lab exercise, we had professional fermenters. At home it would be possible to build one, as I have two air pumps lying around in the basement - just attach a sterile filter.
But I think the cheapest and quickest idea is to keep them in very big containers, with the liquid being just around two centimeters high. Thus free diffusion would be sufficient.
But regulating the pH value without contaminating it will be another issue. Wouldn't it be better to skip pH regulations? Add some Penicillin-G (it should decay during baking) and 8% ethanol. The ethanol will kill most of the bacteria anyway.
The initial pH would be around 5.5, so the yeast has an initial propagation advantage over many bacteria. The ethanol will do the rest.
After some time, the ethanol will be consumed aerobically by the yeast. But it will outgrow any bacteria before they get a chance to propagate I assume.
--
Sent from my Android device with K-9 Mail. Please excuse my brevity.
![Mega [Andreas Stuermer]'s profile photo](http://lh3.googleusercontent.com/a-/ALV-UjUNVm1hB8tAcAAFPeCKG7sAAOMXtiGvFTd7NEBaHFgxRcdy1Lnb=s40-c)
Mega [Andreas Stuermer]
Dec 10, 2013, 12:27:05 PM12/10/13
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Thank you very much!!
I could search the literature whether there extended heat-stable versions of GFP have been characterized. For monellin, they found drastical enhancements in terms of heat stability.
On the other hand, this is mKate (red flouorescent), and it may be more or less stable by default.
I could search the literature whether there extended heat-stable versions of GFP have been characterized. For monellin, they found drastical enhancements in terms of heat stability.
On the other hand, this is mKate (red flouorescent), and it may be more or less stable by default.
Mega [Andreas Stuermer]
Dec 10, 2013, 12:31:05 PM12/10/13
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Cathal Garvey (Phone)
Dec 10, 2013, 2:40:14 PM12/10/13
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Oh, cool!
Nathan McCorkle
Dec 25, 2013, 2:27:44 PM12/25/13
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Andreas Stuermer
Dec 25, 2013, 2:34:06 PM12/25/13
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Wow, nice stuff!
If the GFP-bread itself reaches enough attention, it may be a step further. Synthesizing the Thaumatin gene plus a heat stable GFP. --
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Mega [Andreas Stuermer]
Dec 29, 2013, 1:10:13 PM12/29/13
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http://www.biomedcentral.com/1472-6750/12/65
Fluorescent proteins that work under aneorobic condition and are only 1/10 the lenght of GFP?
Fluorescent proteins that work under aneorobic condition and are only 1/10 the lenght of GFP?
Ideal for fluorescent beer?!?
Mike Horwath
Dec 30, 2013, 10:15:19 AM12/30/13
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Unfortunately, the fluorescence spectra of the tryptophan tags in this article doesn't look so favorable. Excitation maximum ~290nm, Emission max ~340nm. So you are unlikely to see much unless you have UV-B illumination and your eyes can see ultraviolet....
Fluorescent bread/beer is a cool idea! I hope this makes progress!
Mike
Fluorescent bread/beer is a cool idea! I hope this makes progress!
Mike
Mega [Andreas Stuermer]
Dec 30, 2013, 11:35:37 AM12/30/13
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Right. But there will be other proteins that may mature under anaerobic conditions :D
On Tuesday, December 10, 2013 3:32:20 AM UTC+1, Sebastian wrote:
Mega [Andreas Stuermer]
Jan 3, 2014, 1:48:33 PM1/3/14
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http://peds.oxfordjournals.org/content/22/5/313.full.pdf
They put their GFP mutant to 99°C and 30°C repeatedly, and it always recovered again.
Overnight incubation at 80°C didn't harm fluorescence.
How des that look? Can you bake bread at "only" 120°C ? And is there a chance this protein may survive it??
They put their GFP mutant to 99°C and 30°C repeatedly, and it always recovered again.
Overnight incubation at 80°C didn't harm fluorescence.
How des that look? Can you bake bread at "only" 120°C ? And is there a chance this protein may survive it??
Mike Horwath
Jan 5, 2014, 9:36:04 AM1/5/14
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That does look promising!
I've never heard of baking bread at 120C, but I think you could bake hotter. Even in a 200C oven, the interior of the loaf should stay a lot closer to 100C. Turning water to vapor is endothermic and will keep the loaf roughly at boiling temperature. In fact, one way to tell when bread is done is to stick in a thermometer--if the center is at 95C+ it's done.
Another idea could be to try a bagel/pretzel type recipe, where most of the cooking occurs in boiling water. Then just a brief time in the oven to crisp the crust.
Mike
I've never heard of baking bread at 120C, but I think you could bake hotter. Even in a 200C oven, the interior of the loaf should stay a lot closer to 100C. Turning water to vapor is endothermic and will keep the loaf roughly at boiling temperature. In fact, one way to tell when bread is done is to stick in a thermometer--if the center is at 95C+ it's done.
Another idea could be to try a bagel/pretzel type recipe, where most of the cooking occurs in boiling water. Then just a brief time in the oven to crisp the crust.
Mike
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Mega [Andreas Stuermer]
Jan 18, 2014, 11:33:10 AM1/18/14
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As I'm working with mKate instead of GFP:
Is it smart to eat the GM bread? ^^
I mean, the proteins are dentured by baking and also by acid in the stomach.
Thoug prions are subjected to the same procedure and seem to survive well and do damage.
Now, some fluorescent coral protein won't likely act as prions, right? Would you guess your life on it? :P
Is it smart to eat the GM bread? ^^
I mean, the proteins are dentured by baking and also by acid in the stomach.
Thoug prions are subjected to the same procedure and seem to survive well and do damage.
Now, some fluorescent coral protein won't likely act as prions, right? Would you guess your life on it? :P
Matthew Pocock
Jan 18, 2014, 12:45:46 PM1/18/14
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Hi,
I'm not a prions expert, but to the best of my knowledge, GFP and friends are not in any way similar to prions, and should be entirely broken down by digestion - I would expect it to be entirely safe - I've never even heard of anything being allergic to GFP. However, usual disclaimers apply - I've not tested this or seen clinical trials, and wouldn't want to suggest that eating GFP is guaranteed safe or recommend it for any purpose, yada yad yada.
Matthew
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Cathal Garvey
Jan 18, 2014, 1:56:03 PM1/18/14
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Without either endorsing it on legal grounds, nor discounting the
possibility that it *could* do harm (but *only* for stupid legal
reasons), I can safely say that your fluorophore's chances of acting
like a prion are as high as its chances of acting like a perpetual
motion machine.
Prions are endogenous in origin; they refold other versions of
*themselves* in the infected animal, and therefore can only plausibly
emerge from an endogenous protein under as-yet-poorly-understood
circumstances or quirks of evolution.
As you have no proteins that resemble mkate, and as mkate is startlingly
unlikely to be able to misfold other proteins into malevolent form, I
don't see there being any realistic chance of prion-like harm.
More relevant is the question of the fluorophore; is this GFP derived?
In which case the conjugated amino acids that form the fluorophore are
unlikely to be any more harmful than GFP, which appears pretty safe on
ingestion at this point. For some exotic weird fluorophore consisting
entirely of an alkaloid toxin, things would be different. :)
The protein part of your fluorescent protein is likewise unlikely to
pose a serious risk; very few proteins survive digestion, and usually
those have evolved to do so, such as the inflammatory peptides in
gliadans (e.g. gluten) and spore coats or cyst coats that protect parasites.
possibility that it *could* do harm (but *only* for stupid legal
reasons), I can safely say that your fluorophore's chances of acting
like a prion are as high as its chances of acting like a perpetual
motion machine.
Prions are endogenous in origin; they refold other versions of
*themselves* in the infected animal, and therefore can only plausibly
emerge from an endogenous protein under as-yet-poorly-understood
circumstances or quirks of evolution.
As you have no proteins that resemble mkate, and as mkate is startlingly
unlikely to be able to misfold other proteins into malevolent form, I
don't see there being any realistic chance of prion-like harm.
More relevant is the question of the fluorophore; is this GFP derived?
In which case the conjugated amino acids that form the fluorophore are
unlikely to be any more harmful than GFP, which appears pretty safe on
ingestion at this point. For some exotic weird fluorophore consisting
entirely of an alkaloid toxin, things would be different. :)
The protein part of your fluorescent protein is likewise unlikely to
pose a serious risk; very few proteins survive digestion, and usually
those have evolved to do so, such as the inflammatory peptides in
gliadans (e.g. gluten) and spore coats or cyst coats that protect parasites.
DIY BIO Groningen
Jan 18, 2014, 2:30:09 PM1/18/14
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I suggest you to read some related literature about the topic... Here I link you some starting articles.
Is green fluorescent protein toxic to the living cells?
Safety Assessment of Recombinant Green Fluorescent Protein Orally Administered to Weaned Rats
And some background info...
Best,
Alec - DIY BIO Groningen
<0x988B9099.asc>
Simon Quellen Field
Jan 18, 2014, 2:29:59 PM1/18/14
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If all you want is fluorescent bread, why not add turmeric to the dough?
It's a common cheap spice, it tastes good in the bread, it is quite fluorescent, and it survives baking.
You still need a source of ultraviolet light, and eat in the dark, so I'm not sure what the big attraction would be. Putting luminescent bacteria into your yogurt might be more fun, since then you don't need the ultraviolet light. Something like Photobacterium phosphoreum might do nicely -- it has the added benefit of preferring cold environments like the refrigerator, and is not a pathogen. You could add it to the cream cheese you spread on the bread. :-)
Simon Quellen Field
Jan 18, 2014, 2:39:25 PM1/18/14
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Has anyone on this list looked into BioLume?
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Simon Quellen Field
Jan 18, 2014, 2:41:25 PM1/18/14
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Andreas Stuermer
Jan 18, 2014, 3:57:33 PM1/18/14
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Nice.
The GFP study does not apply here, because AFAIK mKate is derived from some coral (and therefore IP free)
The GFP study does not apply here, because AFAIK mKate is derived from some coral (and therefore IP free)
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Simon Quellen Field
Jan 18, 2014, 5:13:31 PM1/18/14
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For $10, you can make your own glowing lollipops using this.
Also good for making ink, hair mousse and cake frosting.
Cathal Garvey
Jan 19, 2014, 5:56:26 AM1/19/14
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I would *definitely* warn off eating Vibrio phosphoreum (née
Photobacterium). I have a paper lying around that found phosphoreum
contained anhydrotetrodotoxin, a precursor to the infamous TTX toxin and
about 25% as toxic as TTX in its own right.
Sadly the paper didn't quantify how much A-TTX was in V.phosphoreum
strains tested, but in the absence of such data it ought to be assumed
unsafe if it contains a neurotoxin in unquantified levels.
Interesting that despite this, V.phosphoreum continues to be classified
as Biohazard Lvl. 1: Essentially, it's safe to work and play with as
long as you don't eat it, and maybe even in that case (but I/we don't know).
Would make a nice project if someone wants to quantify the TTX levels in
this species, provided it's not illegal to work with
toxins-found-in-natural-food-products for the purposes of research in
your area.
On 18/01/14 19:29, Simon Quellen Field wrote:
> If all you want is fluorescent bread, why not add turmeric to the dough?
> It's a common cheap spice, it tastes good in the bread, it is quite
> fluorescent, and it survives baking.
>
> You still need a source of ultraviolet light, and eat in the dark, so I'm
> not sure what the big attraction would be. Putting luminescent bacteria
> into your yogurt might be more fun, since then you don't need the
> ultraviolet light. Something like *Photobacterium phosphoreum
> <http://microbewiki.kenyon.edu/index.php/Photobacterium_phosphoreum>* might
Photobacterium). I have a paper lying around that found phosphoreum
contained anhydrotetrodotoxin, a precursor to the infamous TTX toxin and
about 25% as toxic as TTX in its own right.
Sadly the paper didn't quantify how much A-TTX was in V.phosphoreum
strains tested, but in the absence of such data it ought to be assumed
unsafe if it contains a neurotoxin in unquantified levels.
Interesting that despite this, V.phosphoreum continues to be classified
as Biohazard Lvl. 1: Essentially, it's safe to work and play with as
long as you don't eat it, and maybe even in that case (but I/we don't know).
Would make a nice project if someone wants to quantify the TTX levels in
this species, provided it's not illegal to work with
toxins-found-in-natural-food-products for the purposes of research in
your area.
On 18/01/14 19:29, Simon Quellen Field wrote:
> If all you want is fluorescent bread, why not add turmeric to the dough?
> It's a common cheap spice, it tastes good in the bread, it is quite
> fluorescent, and it survives baking.
>
> You still need a source of ultraviolet light, and eat in the dark, so I'm
> not sure what the big attraction would be. Putting luminescent bacteria
> into your yogurt might be more fun, since then you don't need the
> <http://microbewiki.kenyon.edu/index.php/Photobacterium_phosphoreum>* might
Mega [Andreas Stuermer]
Jan 25, 2014, 1:18:34 PM1/25/14
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Just noticed that my source for the flourescent yeast actually has a very light red, which seems not to be visible to the naked human eye. Does anyone here have access to a fluorescent yeast strain?? Would be very nice!
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