Hacking Your Own Fluorescent Yogurt - Cathal Garvey

[Nathan McCorkle]

Not sure why this hasn't made its way onto the list yet, but its a
great overview!
http://www.indiebiotech.com/?p=152

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[feastduringtheplague]

this is awesome!!!!

On Jan 3, 6:49 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> Not sure why this hasn't made its way onto the list yet, but its a

> great overview!http://www.indiebiotech.com/?p=152

[Ethan]

This is very cool. I was actually going to attempt hacking yogurt as
my first major project when I got my lab up and running. I was
considering bioluminescence. Does anyone know what practical issues
would be a roadblock to producing bioluminescent yogurt? Additionally,
does anyone know anything about toxicity of luciferin or other
intermediates in the bioluminescence pathway that might render it not
such a good idea should the product be consumed?

[Cathal Garvey]

Delighted that this article got a good reception! :) Thanks for
reposting Nathan!

Just an FYI for the general audience: "Luciferin" means "*Any* molecule
that forms the substrate of a bioluminescence system". There are many
luciferins, and they are *not* related at all between systems.

For the most commonly used bacterial luciferin, you're looking at
tetradecanal, an aldehyde taking the form of a 14-carbon chain. It is
found naturally in quite a few food species, but the quantities found in
food species may be far less than those found in bacteria...or more, I
don't know.

Best thing would be to research toxicity of tetradecanal itself, and see
if it's entirely safe. If not, I doubt there's any useful data out there
to prove whether or not the quantities found in a productively
bioluminescent bacteria would be harmful compared to those found in food
plants.

As to "could I make yoghurt bioluminesce", the answer is almost
certainly "yes". *Almost* certainly, with a lot of work, trial and
error. I would strongly suggest starting with something more sure-fire
like GFP, so that you've:
A) Got some experience and
B) Got a positive control to work with when moving onto something more
error-prone like bioluminescence.

If anyone wants to actually pursue this project and hack yoghurt, I'd be
happy to offer advice and such.

By the way, I have in mind a way to do it that would result in an
antibiotic-resistance-free strain at the end: start by replacing the lac
operon using an antibiotic-selection cassette containing your operon,
selecting on glucose medium instead of lactose/milk medium.

Then perform another transformation to replace the antibiotic cassette,
plating on lactose/milk medium to select for transformants. Result;
chromosomal integration of your fluorescence/bioluminescence operon, and
removal of the antibiotic resistance gene. Because your DNA is on the
chromosome rather than a plasmid, it's unlikely to be lost provided
you've ensured there's little if any DNA repetition, and the
evolutionary cost of keeping it isn't punitive.

--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

[Nathan McCorkle]

On Fri, Jan 6, 2012 at 12:31 AM, Ethan <argen...@gmail.com> wrote:
> This is very cool. I was actually going to attempt hacking yogurt as
> my first major project when I got my lab up and running. I was
> considering bioluminescence. Does anyone know what practical issues
> would be a roadblock to producing bioluminescent yogurt? Additionally,
> does anyone know anything about toxicity of luciferin or other
> intermediates in the bioluminescence pathway that might render it not
> such a good idea should the product be consumed?

Regarding toxic stuff, that's something I've thought about before...
Cathal mentions testing the "luciferins" for toxicity, but I think you
might be able to use something like "pathway tools" to do metabolic
analyses... the end products are probably easy to test, but what about
side-reactions that you wouldn't consider. That's what I'd look into
for something like this.

http://bioinformatics.ai.sri.com/ptools/

>
> On Jan 5, 8:23 pm, feastduringtheplague <ntche...@gmail.com> wrote:
>> this is awesome!!!!
>>
>> On Jan 3, 6:49 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
>>
>>
>>
>>
>>
>>
>>
>> > Not sure why this hasn't made its way onto the list yet, but its a
>> > great overview!http://www.indiebiotech.com/?p=152
>>
>> > --
>> > Nathan McCorkle
>> > Rochester Institute of Technology
>> > College of Science, Biotechnology/Bioinformatics
>

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[Patrik]

Firefly luciferin has been used for in vivo imaging:

http://www.invivoimagingcommunity.com/node/74

"at 450mg/kg, no toxicity has been observed [...] Since D-luciferin is
a small molecule it freely diffuses across membranes into and out of
all organs. Luciferin is not metabolized but excreted by the kidneys."
- that seems pretty darn safe.

There's a number of alternative luciferin/luciferase systems as well:

http://www.lifesci.ucsb.edu/~biolum/chem/detail1.html
http://www.lifesci.ucsb.edu/~biolum/chem/detail2.html

As I understand it, the biosynthesis pathway for firefly luciferin is
not known, so you typically need to add luciferin (that then gets
recycled using a separate enzyme). The Vibrio luciferin system is much
better understood though. It's described as "a reduced riboflavin
phosphate" (think vitamin B2...)

On Jan 10, 3:47 pm, Nathan McCorkle <nmz...@gmail.com> wrote:

> > For more options, visit this group athttp://groups.google.com/group/diybio?hl=en.

[Cathal Garvey]

Yes and No. The Vibrio "luciferin" is only half of the system, but
convention dictates that one molecule be called "luciferin" and the rest
are considered cofactors.

The substrates for Vibrio luciferase are the reduced flavin, which is
going to be entirely harmless, and a 14-C aldehyde molecule called
tetradecanal (AKA Myristaldehyde).

I didn't suggest *testing* tetradecanal, I suggested looking it up; it's
almost certainly been tested extensively for toxicity, as it occurs
naturally as an aromatic (as in flavour, not structure) compound in a
number of foods.

..so, that was only slightly harder than I thought it would be to find:
http://www.guidechem.com/dictionary/124-25-4.html

There are tested LD50s and toxicities at the end. It's rated as "Low
toxicity by ingestion and skin contact. A skin irritant."

Toxicity:
1. skn-rbt 500 mg MOD
2. orl-rat LD50:>5 g/kg
3. skn-rbt LD50:>10 g/kg
Source: FCTXAV Food and Cosmetics Toxicology. 11 (1973),487

So, there's your answer; the only significant additives that a *Vibrio*
bioluminescence operon brings are more reduced flavins and tetradecanal,
which seems to be a mild irritant when presented as a pure chemical.

I can't offer advice on whether or not to eat it, but the LD50s are
there for rats, at least; it probably wouldn't kill or even
significantly bother a rat to eat bioluminescent yoghurt. I doubt it
would make a difference to the toxicity at all, for rats. Taste, on the
other hand, it might alter significantly! :)