DIY and open Chymosin (Cheese Hacking) - anyone has some more experience

[Marc Dusseiller]

hei all cheese hackers and genetic engineers,

I was recently looking a bit into cheese making as a metaphor for BioTech, traditional food processing and sharing of starter cultures. It seems to me, that cheese making can also be a great workshop project to foster the discussion on fermentation and micro-biology, aswell as genetic engineering, patent laws, food-labeling and of course being swiss... what else should i do? 

Our recent kimchi workshop experiences has shown a great success in using cooking for introducing people into biohacking, see lab easy, london or share, rijeka.

http://hackteria.org/?p=2616

http://www.artscatalyst.org/experiencelearning/detail/labeasy/

my recent cheese experiments:

http://hackteria.org/?p=2634

The Chymosin, originally extracted from cow's stomach, nowadays is mostly made through recombinant technology, genetic engineering of yeasts or aspergillus niger, to produce the needed enzyme to precipitate (making the curd) the cheese.

http://en.wikipedia.org/wiki/Chymosin#Recombinant_Chymosin

any chance we can circumvent the current patents and make our own plasmid? the open DIY cheese super hiiva? ideally in yeast or something similarly easy to culture?

as this technology is one of the first to be allowed almosts throughout the world, we might be able to engineer our own microbes without too much fuss about legal and safety issues, aswell as social acceptance. unlike some other projects, see glowing plant...

anybody up for it? as i dont really have a clue about plasmid design, i can't really help on that part.

best,

marc

[Cathal Garvey]

Hey Marc,
Would love to work on this, great idea! :)

First question is; how to set up a good antibiotic-free cloning system
for yeast. So, yeast minicircle plasmid with allelopathic toxin,
perhaps? I recall these occur naturally, plasmids that kill cells not
containing the toxin, so they prevent plasmid loss in the population.
Doubt that the toxin is harmful to humans.

And then can just check how they ferment chymosin already and mimic or
improve on that, maybe throw in an affinity tag to make it easy to
purify, and codon optimise for yeast.

I'm currently working on an affinity tag that (if it works) will be
dirt cheap and really easy to use. If it works, I'm looking at lactase
and, now, chymosin as optional side projects along with all the common
lab enzymes. Lab-in-a-box, and fermenting-kit-in-a-box. :)

[John Griessen]

On 08/05/2013 08:27 AM, Cathal Garvey wrote:
> anybody up for it? as i dont really have a clue about plasmid design,
>>i can't really help on that part.

I'm up for the equipment design/sell part of it. It's an idea whose time is now.
Cheese in the store is lame in the US, and there are nice books on making
fancy cheese, but nothing to reduce costs and have a lot of small business activity
delivering local cheeses. and it's a perfect anti-patent area to work in -- old old tech
from the ancients, with a twist.

[Nathan McCorkle]

On Mon, Aug 5, 2013 at 6:27 AM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> Hey Marc,
> Would love to work on this, great idea! :)
>
> First question is; how to set up a good antibiotic-free cloning system
> for yeast.

acetamidase has been used, acetamide is super cheap per kg and if
you're using an integration vector, you can probably get away with
selection less often, so your acetamide should last a while.


--
-Nathan

[Mega [Andreas Sturm]]

I remember a paper saying that the hok/sok system of e.coli also works in yeast too. I assume, if it is toxic to bacteria and yeast, it may be aldo toxic to mammalian cells...

should have a look on pKlac, I guess that's exactly what you'd want...

[jarlemag]

Didn't someone on this list mention some time ago that while toxin/anti-toxin systems indeed prevent plasmid loss, the proportion of transformed cell in culture would still be low, as there's nothing to inhibit the growth of non-transformed cells?  Anyone know more about that?

-JP

[Mega [Andreas Sturm]]

That is true, but a) you could still use acetamide selection
b) attach a signal peptide to the host killing protein so it is secreted in the medium. If other cells take it up (can bacteria take up peptides, or just amino acids?) they die (except they have the surpression of killing protein from the plasmid)

[Tom Randall]

First question is; how to set up a good antibiotic-free cloning system
for yeast. So, yeast minicircle plasmid with allelopathic toxin,
perhaps? I recall these occur naturally, plasmids that kill cells not
containing the toxin, so they prevent plasmid loss in the population.
Doubt that the toxin is harmful to humans.

There is a long history of using auxotrophic mutants for cloning in S. cerevisiae, integrative, centromeric and high copy replicative vectors (URA3 and LEU2, other markers, complementing appropriate mutant strains). This wheel should already have been invented, not sure if there is a central database for these vectors/strains unfortunately but a lot of yeast labs around the world in academia to ask.

[Marc Dusseiller]

Another interesting twist is in the german name for rennet (the enzyme chymosin)...

http://de.wikipedia.org/wiki/Lab

it's called "Lab". So let's try to get these vectors assembled, call it "pOpenLab" and make some cheeeeese.

m

[Cathal Garvey]

You know, concurrent with the launch of SynBio AXLR8R in Cork, Oct.
24th, there is a synbio hackathon planned (precise format yet to be
determined). This could make for a great team project if people are
interested in flying in and hacking it together? :)

At last I heard, the hackathon is no longer tightly associated with the
accelerator itself, such that projects need not be competing for access
to the accelerator, and no onus is on teams to yield a "business-viable"
outcome.

On Thu, 8 Aug 2013 05:00:35 -0700 (PDT)
Marc Dusseiller <dus...@gmail.com> wrote:

> Another interesting twist is in the german name for rennet (the
> enzyme chymosin)...
> http://de.wikipedia.org/wiki/Lab
>

> <https://lh6.googleusercontent.com/-9b9mJ1oiTM0/UgOHuVRx3lI/AAAAAAAAAPo/KPdiAuMuO-g/s1600/IMG_1433.JPG>

[Tim Regan]

Sounds great. I'll be following this..

[Mega [Andreas Sturm]]

Haha :) But you rather pronounce it like Laab, the a pronounced like the one in pizza.

pOpenLaab