Small side project - baking GFP bread - exhibtion

[Mega [Andreas Stuermer]]

Hi everyone,

I am condsidering as a "weekend project" to add GFP to a yeast (saccharomyces, considered GRAS as you know), and then bake bread with it. Haven't talked to the guys from Ars Electronica yet if their Biolab would host this (but I'm positive, if it is legal they will allow it).

Therefore I would subclone GFP into pKlac2 ( https://www.neb.com/~/media/Catalog/All-Products/6F454BB8401043D798342A0719273945/Datacards%20or%20Manuals/N3742Datasheet-Lot0011210.pdf ) fused to the alpha-mating factor which is then cleaved off.

The GFP would then be secreted into the medium (bread dough). So if you cut the dough, there will be small holes which will be fluorescent.

What is questiuonable, will the GFP still work? If it is secreted the yeast will glycosylate it, unlike most cytosolic proteins (according to http://wolfson.huji.ac.il/purification/PDF/Expression_Systems/NEB_Klactiskit_manual.pdf )
Should I express it in the cytosol - no golgi etc.?


Thanks,
Andreas

[Mega [Andreas Stuermer]]

P.S. I have considered that baking will destroy GFP anyway. But the raw dough will make beautiful fotos :D

[Cathal Garvey (Phone)]

I'm more concerned that there won't be time during rising for yeast to make enough GFP: have you considered a sourdough instead? Coculture bacteria and yeast, long fermentatiob, plenty of time for GFP?

--
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[Nathan McCorkle]

I support any pklac2-based projects!

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--
-Nathan

[Mega [Andreas Stuermer]]

I'm more concerned that there won't be time during rising for yeast to make enough GFP: have you considered a sourdough instead? Coculture bacteria and yeast, long fermentatiob, plenty of time for GFP?

Really? I think we said with GFP beer that GFPs don't work in acidic environments. But it was also due to need of oxygen for properly folding.

GFP needs I think some 20 mins to fold properly (IIRC).

I am reading what amount of time the yeast usually works, one page mentioned 1 hour. Maybe I need to sit it for 2.5 hours to get enough GFP? The taste of the final breadf is not that important, though I would like to eat it still.



Nathan, ;)
I love the acetamidase selection marker. Will be nice to yield "green" GMOs :)

[Mega [Andreas Stuermer]]

If I add some glucose/sucrose to the dough, that will speed up the yeast.

"

Secreted proteins may also bear post-translational

modifications (e.g. asparagine-linked glycosylation) that can be removed by

treatment with Endo H (NEB #P0702) or PNGase F (NEB #P0704)"

"Secretion of a protein of interest from K. lactis cells is the most common

approach to protein expression. Secretion results in production of proteins that

are significantly pure, that do not require difficult lysis of yeast cells to isolate,

and that may have desired post-translational modifications (e.g. glycosylation)

that cytosolic proteins do not"

In jellyfish it will be glycsyllated too? Doesn't s cerevisiae tend to overglycosyllate? Will it get unfunctional?

.

[Mega [Andreas Stuermer]]

http://www.ncbi.nlm.nih.gov/pubmed/15801770

Seems it has already been done, to secrete GFP from yeast cells.


I would love the yeast to secrete GFP, though it is less likely to work then^^
If I just express it in the cytosol, that probably makes more sense...? However, Kozak sequences are more difficult to design than SD... Have no hint if it will work like that :D

[Mega [Andreas Stuermer]]

Budding yeast (Saccharomyces cerevisiae)

Ascomycota

aAaAaAATGTCt That's the Kozak. Accidentally, the second codon(TCt) is the same as the second codon in GFP!

Both options are nice, but which one to take??

[Nathan McCorkle]

I was previously advised that if I wanted to augment pklac2 with
another promoter, that I could use about 300 bp of DNA upstream of the
ATG start of the PGK1 gene. The PGK1 promoter can be isolated from the
following S. cerevisiae site:
http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=PGK1

So I imagine what you want is buried in there... somewhere

[Koeng]

I couldn't find the sequence online, but the lab I work at has a plasmid that holds mKATE, like RFP, that is easily expressible in yeast... (One of the grad students even had his plates infected by yeast with the integration because they spored)

[Mega [Andreas Stuermer]]

Another promoter won't be neccesary I think? If I insert it into HindIII site, the secretion signal peptide is cleaved off and it stays in the cytoplasm.

[Mega [Andreas Stuermer]]

By the way, we can do a survey which fluorescent protein I'll use :D 

Green looks nice, but so does orange or red. Blue or yellow bread would look kind of wired... :P 

On Sunday, November 10, 2013 11:29:09 AM UTC+1, Mega [Andreas Stuermer] wrote:

[Mega [Andreas Stuermer]]

I may be able to get a ready-to use fluorescent protein yeast integrative plasmid, rather than need to cut GFP into pKlac2.

However, the ready-to -use plasmid often either contain

a) URA marker, so you need a URA-deficient strain of backers yeast -> cost, time, where to get it from?
b) antibiotic markers - chloramphenicol, kanamycin, etc.


Although I always tell everyone I know that it is very unlikely that the antibiotic gene gets into your gut bacteria, and chances are even smaller that they can even express it... I would have a bad gut feeling of eating yeast with a resistance gene... Is it just superstitious me, or is there some truth in it?

The yeast DNA will survive baking very well, so in theory it should be possible that bacteria take up a fragment. But without a SD-type RBS...?


Would you eat it?

[Nathan McCorkle]

Horizontal transfer exists, maybe search for GI (gastrointestinal
tract) species horizontal transfer?

I wouldn't worry about antibiotics not commonly used (or not approved)
in humans, though they might still be used as last-resort measure for
really sick people... it probably doesn't happen much because you'd
probably have to be in really really bad shape to get your doctor to
give you unapproved/blacklisted medication. That said, you should
avoid non-approved drugs that might impact the pressure on some of the
approved-for-humans antibiotic mechanisms.

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[Koeng]

There isn't really any selection for antibiotic resistance in your gut. DNA transformation is inefficient even with a bunch of stuff we do to make them competent. I wouldn't worry about it

-Koeng

[Cathal Garvey]

Citation needed. DNA transfer by conjugation is *very efficient*, and
many species found abundantly in the gut and environment are naturally
competent.

I'll grant you, there are few species in the gut that produce medical
antibiotics (streptomyces etc.).

If this is a once-off thing, don't worry about it (but you're still
forgetting that you're *not allowed eat it in Europe* so it's
technically irrelevant). If people will be keeping and eating this
elsewhere, perhaps do something different and "food-grade":

Get the Nisin immunity gene operon, ~3 genes IIRC, and buy some
food-grade nisin, used industrially as a food-grade preservative. Not
used medically, digestible without side effects, etc.

If you're using Yeast, don't even bother; use an integrative plasmid,
and just grow normally. Yeast already produces an effective
antibacterial toxin; alcohol! If fermented in sourdough, the alcohol is
made into acid instead, which has the same net result; only Yeast and
(moreso) lactic acid / acetobacter bacteria thrive.

I feel that antibiotics are for medicine and veterinary use. I think if
you agree, you ought to make an effort to avoid them when you can
afford to, and minimise them when you can't.

[Nathan McCorkle]

On Tue, Nov 12, 2013 at 5:51 AM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> Get the Nisin immunity gene operon, ~3 genes IIRC, and buy some
> food-grade nisin, used industrially as a food-grade preservative. Not
> used medically, digestible without side effects, etc.

...

> I feel that antibiotics are for medicine and veterinary use. I think if
> you agree, you ought to make an effort to avoid them when you can
> afford to, and minimise them when you can't.

Couldn't one say that using nisin resistance could lead to increased
food spoilage proliferation, which wouldn't be as bad as MRSA, but
would still impact humans health and well-being.

--
-Nathan

[Meredith L. Patterson]

What about cadmium resistance? I've seen that referenced in papers on food-grade lactic acid bacteria.

Cheers,

--mlp

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[Mega [Andreas Stuermer]]

But where to get the cadmium. How not to get sick handling it. And how to discard it properly?

Yellow street markings contained cadmium quite some time ago... Do the soil around streets is often contaminated with cadmium. Purify it from there? ^^

[Meredith L. Patterson]

Cadmium's easy to get -- $12 for 200g on eBay (http://www.ebay.com/itm/Cadmium-metal-rod-200g-99-98-Kadmium-Metall-pure-element-Cadmio-/221282636722?pt=LH_DefaultDomain_0&hash=item33857913b2) -- but yeah, handling is an issue.

Cheers,

--mlp

On Wed, Nov 13, 2013 at 8:04 AM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

But where to get the cadmium. How not to get sick handling it. And how to discard it properly?

Yellow street markings contained cadmium quite some time ago... Do the soil around streets is often contaminated with cadmium. Purify it from there? ^^

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[Cathal Garvey (Phone)]

Really? :) Food spoilage isn't even on the same spectrum here. But even so, nisin's in effectively uncontrolled use and widespread use at that. Resistance is likely widespread already.

That said, if you really feel like a model citizen you could avoid plasmids known to function well in food pathogens or known spoilage bacteria. The former includes S.Aureus, the latter B.subtilis, but IIRC those two are BFFs with lactobacilli so it'd be an interesting fringe challenge.

For the record I'm not a fan of this cadmium idea!

[Eugen Leitl]

On Wed, Nov 13, 2013 at 09:03:53AM +0100, Meredith L. Patterson wrote:
> Cadmium's easy to get -- $12 for 200g on eBay (
> http://www.ebay.com/itm/Cadmium-metal-rod-200g-99-98-Kadmium-Metall-pure-element-Cadmio-/221282636722?pt=LH_DefaultDomain_0&hash=item33857913b2)
> -- but yeah, handling is an issue.

Cadmium is definitely not mercury, so handle it
the same way you would handle lead. Don't ingest it,
wash your hands, dispose of properly.

[Mega [Andreas Stuermer]]

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.