Requesting your thoughts on a yogurt-based nutraceutical platform
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Richard Yu
Oct 3, 2013, 1:00:39 PM10/3/13
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Hi DIYbio,
I know many of you have thought quite a bit about hacking yogurt (L. bulgaricus, specifically), and I wanted to solicit your thoughts on a "20% time" fun project some friends of mine and I are pursuing at a natural products biotech startup (http://igg.me/at/yovivo)
-Frameworks:
We've chosen resveratrol yogurt as a POC/test project. We are actually serious about bringing this to food production stages, so in addition to the usual difficulties of biology of just getting it to work period, there are other issues we need to consider- FDA rules/regs surrounding eating a microbial GMO product, and how to actively engage with other communities that may not be so sympathetic to GMOs. Sure, many of us have a dream of having a milk-based platform for making whatever nutraceutical or med or glowing/fluorescent protein or smell or flavor- what are your concerns about such a platform? what do you think the general public's concerns might be?
-Projects:
Do you guys have thoughts on specific related projects that might be fun and important? This includes other products (we've gotten about a billion requests for frozen yogurt) or even fun projects we might do to engage with the DIYbio community, perhaps at a space like Biocurious? What are informative things that we can do, and how can we do them, so that won't needlessly fan the flames of anti-GMO sentiment and make our work and play harder?
Thanks in advance for any thoughts!
Best,
Rich
Meredith L. Patterson
Oct 4, 2013, 9:33:37 AM10/4/13
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Hi Richard,
Cheers,
I saw your Indiegogo the other day and was planning on getting in touch with you -- great to see you here!
A couple of iGEM students here in Brussels and I decided a few weeks ago to reboot the melaminometer project (openwetware.org/wiki/User:Jonathan_Cline/Notebook/Melaminometer), and we're in the process of rebuilding my lab (we're doing inventory as we speak, actually). My pipe-dream project, though, is to produce a strain of lactic acid bacteria that expresses L-gulonolactone oxidase, the last enzymatic step in the mammalian ascorbate synthesis pathway (the final step is spontaneous). Humans and guinea pigs only have a pseudogene for this enzyme, and so we can't make our own vitamin C. I'm not sure whether expression of GULO in the gut will restart the pathway (if not, then the next question is whether one of the full ascorbate synthesis pathways can be packed onto a plasmid), but that's the idea that got me into DIYbio in the first place.
What can you tell us about the plasmids you're using?
Cheers,
--mlp
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Richard Yu
Oct 4, 2013, 12:47:56 PM10/4/13
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Hi MLP-
Thanks for reachout! We're going to start using the "standard" plasmids that people have had the best luck with in the lit- pJK650, pLEM415, and pX3. Just to get the bugs to make anything. Though, these days with Crispr and everything, the idea of high efficieincy markerless homologous recombination into a predefined neutral locus would be ideal. We're focused on using food-grade DNA parts, so no antibiotic resistance markers anyway, but there's something very elegant about the markerless homologous recombination!
I love the idea of both the melamine sensor and the vitamin C! We were considering a beta-carotene project, ("Golden Yogurt?"), but for a variety of reasons decided that resveratrol was both an interesting base pathway, and it would let us hit some synbio territory that hadn't been covered. (We are completely aware of all the work to heterologously produce resveratrol in microbes, as well as standing on the shoulders of both resveratrol beer (many) and Igem2008 Biogurt. We try to give props every step of the way!) Seems like the GULO experiment is straightforward enough to test. Would be interested to keep in touch and hear how it progresses!
Cheers,
Rich
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Mega [Andreas Sturm]
Oct 5, 2013, 5:40:37 AM10/5/13
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Hi!
Will you share the plasmids as a pledge?^^
Resveratrol is great!
What would also be kind of nice is the bacterial lux pathway. It is fatty aldehydes (present in human body), luciferase (proteins, won't be toxic), and some activated vitamin B2.
If you open the yoghurt, oxygen comes and activates the glowing. :D
But I read you would sterilize the yoghurt to ensure GMO containment?
I personally would love to eat GM-enhanced yoghurt, especailly resveratrol because I don't drink vine and my body is out of resveratrol :D
Will you share the plasmids as a pledge?^^
Resveratrol is great!
What would also be kind of nice is the bacterial lux pathway. It is fatty aldehydes (present in human body), luciferase (proteins, won't be toxic), and some activated vitamin B2.
If you open the yoghurt, oxygen comes and activates the glowing. :D
But I read you would sterilize the yoghurt to ensure GMO containment?
I personally would love to eat GM-enhanced yoghurt, especailly resveratrol because I don't drink vine and my body is out of resveratrol :D
Mega [Andreas Sturm]
Oct 5, 2013, 5:45:07 AM10/5/13
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There is a protein called monellin. It is said to be much, much sweeter than sugar.
There is also a modified version which is thermostable and surivives 100°C (re-folds correctly again).
Would be a nice calorie restricted, still sweet yoghurt.
http://openwetware.org/wiki/IGEM:MIT/2006/Blurb
Make sure to check out this. You can also give it the taste of banana. Or wintergreen.
Definitely would taste nice!
There is also a modified version which is thermostable and surivives 100°C (re-folds correctly again).
Would be a nice calorie restricted, still sweet yoghurt.
http://openwetware.org/wiki/IGEM:MIT/2006/Blurb
Make sure to check out this. You can also give it the taste of banana. Or wintergreen.
Definitely would taste nice!
On Thursday, October 3, 2013 7:00:39 PM UTC+2, Richard Yu wrote:
A
Mega [Andreas Stuermer]
Oct 5, 2013, 5:54:09 AM10/5/13
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mlp,
L-gulonolactone oxidase is an awesome idea. I was looking forward to the day when gene therapy for this is legal ( :D ), but would expressing it in gut microbes is a great idea too. However, won't the protein be too big to penetrate the gut epithel? I think so, unfortunately...
L-gulonolactone oxidase is an awesome idea. I was looking forward to the day when gene therapy for this is legal ( :D ), but would expressing it in gut microbes is a great idea too. However, won't the protein be too big to penetrate the gut epithel? I think so, unfortunately...
On Thursday, October 3, 2013 7:00:39 PM UTC+2, Richard Yu wrote:
Cathal Garvey (Phone)
Oct 5, 2013, 6:23:20 AM10/5/13
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I've already suggested plasmids-as-pledge or optional live cultures. At present they are only planning to release pasteurised yogurt making thus proprietary.
I'll support it when it's A) Live and B) Free/Libre; no patents!
As far as resveratrol, it's also found in peanuts, and it's abundant in japanese knotweed, an invasive weed in much of Europe. The capsules you buy are knotweed, mostly. Bear in mind though that intestinal absorption is negligible, so oral cavity absorption is probably key..and as it's poorly water soluble, alcohol is probably necessary. I take mine by breaking capsule into a shot of cheap red wine and swishing it around!
The lactic acid in yogurt and the tiny bit of alcohol that tends to creep in might help absorption from yogurt.
I'll support it when it's A) Live and B) Free/Libre; no patents!
As far as resveratrol, it's also found in peanuts, and it's abundant in japanese knotweed, an invasive weed in much of Europe. The capsules you buy are knotweed, mostly. Bear in mind though that intestinal absorption is negligible, so oral cavity absorption is probably key..and as it's poorly water soluble, alcohol is probably necessary. I take mine by breaking capsule into a shot of cheap red wine and swishing it around!
The lactic acid in yogurt and the tiny bit of alcohol that tends to creep in might help absorption from yogurt.
--
Sent from my Android device with K-9 Mail. Please excuse my brevity.
Sent from my Android device with K-9 Mail. Please excuse my brevity.
Mega [Andreas Stuermer]
Oct 5, 2013, 8:09:18 AM10/5/13
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Oh, cool. I think I've already seen that weed.
However, the wiki article says it contains oxalic acids, which makes kidney stones. My father hade stones, quite painful. I think I rather avoid it...
However, the wiki article says it contains oxalic acids, which makes kidney stones. My father hade stones, quite painful. I think I rather avoid it...
On Thursday, October 3, 2013 7:00:39 PM UTC+2, Richard Yu wrote:
Nathan McCorkle
Oct 5, 2013, 2:21:18 PM10/5/13
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On Oct 5, 2013 2:45 AM, "Mega [Andreas Sturm]" <masters...@gmail.com> wrote:
>
> There is a protein called monellin. It is said to be much, much sweeter than sugar.
I heard a talk by Cynthia kenyon on c elegans longevity research and it showed that even olfactory stimulation by sweet stuff caused an expression response that could eventually lead to similar insulin resistance scenarios (type 2 diabetes).
So it seems moderation is the key to anything sweet regardless of caloric density. That said, seems sugar is a simpler solution if non-calorie sweeteners don't actually taste more /interesting/ than common sugars. Things like miraculin are cool, sugar substitutes aren't nearly as exciting.
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Richard Yu
Oct 5, 2013, 7:29:28 PM10/5/13
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Hi all,
Thanks for the discussion everyone!
We're looking into what's the best way to release something live- live yogurt, or a kit that you can transform in a lab (lacto + plasmids), or something else. Though we're actually interested in making this a product for general sale, hence some of the "err on the side of safety while things are getting worked out" things (like pasteurization before general release), I personally understand the thing that psyches the community of doers and makers here is the ability to open and play and break and fix. (As someone who will spend a whole day and 10x the cost in parts to fix a $19 Target toaster oven just on principle- we do it because we are compelled). I am intrigued by the idea of plasmids as a perk for a pledge; I'll definitely pitch that at our next meeting.
Proprietary may be an incorrect way to cast the "sterilization" angle; I think it's more "containment at the source". I don't personally have a problem distributing the DNA sequence of the plasmids we build, for example.
Part of the money we're raising is to deal with these issues and to work with legal people and regulators to actively shape what's going on about how to release GMOs or parts to make GMOs. So, we're wanting to do this, we just want to do it right. I'm all for taking a principled stand, but I don't want to be responsible for catalyzing or causing an overcompensating backlash that hurts or kills this community. And input form you guys about ways to do that is awesome.
So, what do you think? Antony Evans (of Glowingplant fame) suggested kits that are sent to lab addresses, with proper containment and toxic substance licenses/permits (I know, it's simply 10% bleach in a bucket). What else can we do to get closer to what Cathal and Andreas would like?
As for other products- flavors, sweeteners, and luciferase- all cool. Miraculin is fun/funny too.
Thanks again for the conversation, would love to see where this goes!
Best all,
Rich
Nathan McCorkle
Oct 6, 2013, 1:41:33 AM10/6/13
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I think there was talk a while ago about getting pVIB to work... light
emitting yogurt would definitely be cool.
emitting yogurt would definitely be cool.
Nathan McCorkle
Oct 6, 2013, 3:12:30 AM10/6/13
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On Sat, Oct 5, 2013 at 4:29 PM, Richard Yu <r...@radiantgenomics.com> wrote:
> We're looking into what's the best way to release something live- live
> yogurt, or a kit that you can transform in a lab (lacto + plasmids), or
> something else.
> We're looking into what's the best way to release something live- live
> yogurt, or a kit that you can transform in a lab (lacto + plasmids), or
> something else.
> I am
> intrigued by the idea of plasmids as a perk for a pledge; I'll definitely
> pitch that at our next meeting.
With provide-your-own yogurt and centrifuge, providing a plasmid could
> intrigued by the idea of plasmids as a perk for a pledge; I'll definitely
> pitch that at our next meeting.
be a proof-of-concept kit! I've been working on building an
electroporator for a while, last used e.coli and a grill lighter piezo
unsuccessfully, but there are a bunch of papers on l. delbrueckii
getting zapped that I'd be excited to try! Do you have any of the
shuttle vectors now?
None of them really mention using milk agar though, mostly MRS media
which is somewhat expensive at $160 for 500g powder.
Meredith L. Patterson
Oct 6, 2013, 6:25:06 AM10/6/13
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Make your own Elliker medium! Here's the broth composition that I got out of the Journal of Dairy Sciences from 1956:
Tryptone 2%
Yeast extract 0.5%
Gelatin .25%
Dextrose .5%
Lactose .5%
Sucrose .5%
Sodium chloride .4%
Sodium acetate .15%
Ascorbic acid .05%
All percentages are w/v; if you're going to pour plates, add 1.5% agar. See also http://www.jsunitech.com/product/culture/manual/Elliker%20Broth.pdf, which gives measurements in SI units (5g yeast extract, 2.5g gelatin, &c), and swaps in casein peptone as a replacement for tryptone.
Cathal, whatever happened to your casein peptone protocol? I can't find it on your blog anymore :(
According to http://www.scribd.com/doc/24840826/2010-Lactic-Acid-Bacteria-Potential-for-Control-of-Mould-Growth-And, Elliker medium is actually better than MRS broth for production of antifungal compounds, for what that's worth.
Cheers,
--mlp
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Andreas Sturm
Oct 6, 2013, 6:31:13 AM10/6/13
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As for pVIB, in E. Coli it works just awesome.
Ah, lactobacillus is gram positive... So pVIB won't work well with that. Once there was research done which added gram positive RBS and a bacillus promoter to the lux operon.
Ah, lactobacillus is gram positive... So pVIB won't work well with that. Once there was research done which added gram positive RBS and a bacillus promoter to the lux operon.
I believe your idea could make GMOs more acceptable.
People see your yoghurt and it says "makes you live longer and healthier" and "contains GMO derived products". Then they have to choose. And even the most (pseudo-)environmentalist wants to live a longer healthier life...
So, many people will choose GMO if it brings health benefits (instead of making it insecticide producing)
On Sun, Oct 6, 2013 at 9:12 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
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Cathal Garvey (Phone)
Oct 6, 2013, 6:50:05 AM10/6/13
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..even if said insecticide counts as a nutritious source of digestible protein, in humans.
Andreas Sturm
Oct 6, 2013, 7:16:14 AM10/6/13
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There was a paper, which descirbed the construction of a grm-positive lux vector and even revealed the sequence...
Can't find the article anymore, but if I can send you for future projects...
Similar ones...
There was also an old one for B. Subtilis, which glowed noticable by naked eye when carriying the high-copy number plasmid. But when a single-copy was integrated into the genome, it glowed too weak to see.
Richard Yu
Oct 6, 2013, 5:43:27 PM10/6/13
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Hi Andreas,
On Sunday, October 6, 2013 3:31:13 AM UTC-7, Mega [Andreas Stuermer] wrote.
I certainly hope that it helps, at the very least, create some constructive dialogue and perhaps open some previously closed minds.
I want to see realized the potential of this for enabling the poor to make their own meds, meds that are not being produced for the usual economic reasons. I hope that that is something most people can consider a worthy goal, despite the large amount of work it will take to get there safely.
As for promoters, we're planning on using native constiutive/strong Lactobacillus promoters, might as well stay food-grade an not go heterologous. We're also using only genes from red grapes. Addresses the potential "allergy" issue of using insect (or other foreign) proteins.
A humble request:
If you guys like what you see, please follow/tweet about us @yivivoyogurt and like our page on Facebook https://www.facebook.com/yovivoyogurt ? WE're in the early stages of our campaign and could really use the early momentum and support! (Contributions would be awesome, too! I'm working on the live/kit thing ASAP!)
Thanks all! I really appreciating the genuine psyche and interest in DIYbio. Hope to keep the conversation going.
Rich
On Sunday, October 6, 2013 3:31:13 AM UTC-7, Mega [Andreas Stuermer] wrote.
[...]
I believe your idea could make GMOs more acceptable.People see your yoghurt and it says "makes you live longer and healthier" and "contains GMO derived products". Then they have to choose. And even the most (pseudo-)environmentalist wants to live a longer healthier life...
So, many people will choose GMO if it brings health benefits (instead of making it insecticide producing)
[...]
Richard Yu
Oct 7, 2013, 4:47:40 PM10/7/13
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Hi all,
I just wanted to let you know that after many discussions we're offering DIY kits as perks!
I'd like your input on them. For now, we're discussing distributing kits that have:
-Lactobacillus streak or stab
-premade plasmid to transform (or empty vector if you choose)
-a few plates for a couple tries at recovery
Now, with Lacto, you'll need an electroporator for now. But if some enterprising enthusiast can figure out a chemical transformation procedure, that'd be most rad (we'll be trying some stuff).
We'll be thinking about some of the plasmids we can make and offer, including some your excellent suggestions here.
It took a while to get here- this is a new thing for us, and we're trying to get there as fast as we can. (clearly not professional marketing people here...) We appreciate your patience!
Please share if you like it! http://igg.me/at/yovivo
Best,
Rich
Cathal Garvey (Phone)
Oct 7, 2013, 5:25:04 PM10/7/13
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Hey Richard,
That's brilliant news, thanks to you and your team! Will be delighted to contribute and advocate for an open, hackable yoghurt! :)
That's brilliant news, thanks to you and your team! Will be delighted to contribute and advocate for an open, hackable yoghurt! :)
Josiah Zayner
Oct 7, 2013, 5:44:13 PM10/7/13
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There is already a patent for this process: http://www.google.com/patents/WO2006124999A3?cl=en
Though I don't know how much that matters. The glowing plant project was selling someone else's patented idea.
Resveratrol from what I see can be purchased for really cheap 15-30g for $11 or so. How does your system make it any better than just me adding resveratrol to my yogurt?
What is the estimate in the amount of resveratrol that can be produced in say 200mL of yogurt? The paper you put in your Indiegogo says 16mg/L in E. coli(probably much lower for a wildtype Lacto). That is more than wine but at that amount(3mg/200mL) I could pay $11 and have >20L of resveratrol laden yogurt.
Your Indiegogo says 10x the amount found in wine. From what the literature says, taking a median value, there is currently around a 4 fold difference (16mg/L, E. coli and 1-7mg/L Wine). How do you plan to come up with the other ~6 fold? Or are you using the lower estimates of resveratrol in glasses of wine? In the paper you cite on your Indiegogo for increased resveratrol production they are engineering yeast and though they say they have a 2.44 fold higher resveratrol production it is still only ~3mg/L total. The other papers I have seen on higher resveratrol production are in yeast also.
So do you have any idea how you will increase this production? Or if the system will even work in Lacto?
Richard Yu
Oct 7, 2013, 6:07:38 PM10/7/13
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Hi Josiah,
Nope, not claiming to do anything new here with regard to microbial production of resveratrol; that's why we referenced the papers. It's more about marrying the concept with a food-based microbial platform, and using resveratrol as a nice pilot project.
As with most of these things, you might not buy it in one format or another to save money. Even if you pay for micronized resveratrol supplements, you'd probably break even with eating a pill and buying plain yogurt. (Your statements above don't factor in the cost of >20L of yogurt, but I get your point). We'll see how competitively we can price it.
I don't know about absorption advantages in the form we propose- we will have to see! There may be benefits, maybe not. My guess is it at least won't be worse!
As for lacto engineering, the lit is a bit sparse, as it's not really a dominant microbial platform. So a lot of this, we will have to see! Biology being what it is, there are no guarantees.
We picked 10fold assuming 3 mg/L wine because at around 30 mg/L you start seeing more of the benefits beyond cardiovascular health (table 2, Mukherjee et al 2010). The papers I saw maxed out at 5.3 mg/L for pinot noir (Lamuela-Raventos et al 1995). Assuming we take it from there, 16 mg/L for ecoli is ~4-5 fold higher. the other 2-3 fold (not 6 fold) to get to 10 fold is something we as metabolic engineers are hopeful we can attain. It's not a gimme- as you point out, E.coli is likely to be better than native Lacto (though you never know). But not totally out of the park for guys who've increased things many orders of magnitude before.
Thanks for the input! Hope that answers your questions,
Rich
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Patrik D'haeseleer
Oct 7, 2013, 10:50:54 PM10/7/13
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Very cool project! I'd love it if you guys could come give a talk about your work at Counter Culture Labs - your friendly neighborhood DIYbio group. You're right in our backyard after all! This is just the kind of projects we'd love to get into once we get our lab up and running.
Do you foresee any trouble synthesizing the p-coumaric acid in lacto? The high-throughput E. coli resveratrol biosynthesis papers I've seen (e.g.
High-Yield Resveratrol Production in Engineered Escherichia coli - 2.3 g/liter resveratrol!) all seem to feed in p-coumaric acid, because getting the C4H enzyme to work in bacteria seems problematic:
Patrik
Do you foresee any trouble synthesizing the p-coumaric acid in lacto? The high-throughput E. coli resveratrol biosynthesis papers I've seen (e.g.
High-Yield Resveratrol Production in Engineered Escherichia coli - 2.3 g/liter resveratrol!) all seem to feed in p-coumaric acid, because getting the C4H enzyme to work in bacteria seems problematic:
One of the barriers to the production of flavonoids and their related compounds in microorganisms by means of assembling biosynthetic genes to form an artificial pathway is the difficulty in expression of active, membrane-bound C4H. This enzyme is not expressed efficiently in bacteria due to its instability and the lack of its cognate cytochrome P450 reductase in the host.
Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms
Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms
Patrik
Richard Yu
Oct 7, 2013, 11:40:52 PM10/7/13
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Hi Patrick-
Thanks so much for your support!
Good points. I don't know how Lacto will differ from Ecoli in terms of expresion of active C4H, or what the flux will be like in Lacto. We're hoping to stick with the simple things- expression levels, perhaps protein fusions might also do the trick (analogous to (Zhang, Y et al, JACS 128:13030-13031 (2006))). Fusing PAL to C4H, or C4h to 4CL? Get that substrate channeling thing going. The farther we get away from native proteins, though, the farther we get away from that food-grade criteria for a food product. It's an interesting set of scientific and non-scientific constraints to match :). Will be fun to see how far we can bring standard metabolic engineering and strain improvement techniques into the fold, too. (mutagenesis and selection, for example- not GMO!). For specifics of flux limitation, we will have to get our hands wet and start measuring!
As for your question about electroporation on Twitter, I'm going by what my friends in the BioGurt 2008 MIT iGEM team mentioned, though Jeff did mention some other transformation protocols. I've read Cathal's idea to use S. thermo. w/ Competence pheromone for chemical transformation.
Might be time so search for something that might work for Lacto. More on this later, for sure!
Best,
Rich
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/23cf816c-35e1-4e1f-af06-c4e2f51a454c%40googlegroups.com.
Richard Yu
Oct 8, 2013, 2:05:26 AM10/8/13
to diybio
Hey Patrick,
We were just reading about alternatives to electroporation and we found of some methods that might work- this is analogous to techniques I heard about for Chlamy back in my biofuel days, where vortexing cells, DNA, and glass beads or silicon whiskers could transform pretty efficiently. Jeff found a paper that describes another fibrous matrix-
http://www.deepdyve.com/lp/elsevier/a-simple-and-rapid-method-of-bacterial-transformation-qYLXb2Jcjo
Buffered kitty litter. Huh, who'da thought. Throw in some PEG and vortex and you're probably in business.
Rich
ps. changed the perks to reflect that; yours may not be up there anymore but rest assured you're getting your kit if we break 25K!
Nathan McCorkle
Oct 8, 2013, 3:06:15 AM10/8/13
to diybio
On Mon, Oct 7, 2013 at 11:05 PM, Richard Yu <r...@radiantgenomics.com> wrote:
> ps. changed the perks to reflect that; yours may not be up there anymore but
> rest assured you're getting your kit if we break 25K!
$200 for a scoop of yogurt and a plasmid? I thought a plasmid would be more like
> ps. changed the perks to reflect that; yours may not be up there anymore but
> rest assured you're getting your kit if we break 25K!
$20 and plasmid+stab $50 max.
Richard Yu
Oct 8, 2013, 3:19:53 AM10/8/13
to diybio
I will talk pricing over with my teammates tomorrow. We're trying to balance time+effort, material costs, and embedded costs with the price. On the one hand, things can't be ridiculously expensive; on the other hand, this isn't a discount store, it's meant to be fundraising for a project that inspires and excites you- we got a lot of work to do, and it ain't cheap!. ATCC will charge $50-60 for the strain, usually at least $50 for a plasmid. and often hundreds of dollars for a plasmid.
But would like to hear other's opinions- too much? Glowing Plant charged $250 for their maker kit, for a one point reference.
Best,
Rich
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Nathan McCorkle
Oct 8, 2013, 3:33:14 AM10/8/13
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Carolina.com sells e.coli ampicillin and a plasmid for $24, presumably plus shipping.
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Mega [Andreas Stuermer]
Oct 8, 2013, 3:54:05 AM10/8/13
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Cool that diy kits are now available... Unfortunately shipment only to US :(
As for the diy kit, it should be something that people can sense on their own, like fluorescence or smell :D Maybe even the colour? Avery has a gene that produces colour, but don't know if that works outside of E.Coli. And btw, who would eat a blue youghurt .D
Easy to accomplish for the perk would be GFP.
More fascinating lux, but gotta get a gram-prositive version. Plus, you could say lux produces activated vitamin B2, thus it is healthy to eat glowing yoghurt :D
Nice would be having it taste and smell like wintergreen, but this requires 2 genes, IIRC like 2000 bp and synthesizing this will be in the range of 800$.
As for the diy kit, it should be something that people can sense on their own, like fluorescence or smell :D Maybe even the colour? Avery has a gene that produces colour, but don't know if that works outside of E.Coli. And btw, who would eat a blue youghurt .D
Easy to accomplish for the perk would be GFP.
More fascinating lux, but gotta get a gram-prositive version. Plus, you could say lux produces activated vitamin B2, thus it is healthy to eat glowing yoghurt :D
Nice would be having it taste and smell like wintergreen, but this requires 2 genes, IIRC like 2000 bp and synthesizing this will be in the range of 800$.
Mega [Andreas Stuermer]
Oct 8, 2013, 4:01:50 AM10/8/13
to diy...@googlegroups.com
here on this list someone pointed out a homepage that selled plamids ,
and there were two plasmids, lux for gram positive and lux for gram negative.
http://www.perkinelmer.com/Catalog/Product/ID/119253
It was not this I think, but here's a g.p. lux
and there were two plasmids, lux for gram positive and lux for gram negative.
http://www.perkinelmer.com/Catalog/Product/ID/119253
It was not this I think, but here's a g.p. lux
Patrik D'haeseleer
Oct 8, 2013, 4:02:48 AM10/8/13
to diy...@googlegroups.com
Rule of thumb for kickstarter is that people spend around 1/3 of the money they raise on fulfilling rewards. So figure out how cheap you can produce the kits, make sure to account for packing and shipping, and then multiply by three.
Mega [Andreas Stuermer]
Oct 8, 2013, 4:07:36 AM10/8/13
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Mega [Andreas Stuermer]
Oct 8, 2013, 4:13:04 AM10/8/13
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If they don't sell anymore, here's a chinese page that sells. http://www.summitpharma.co.jp/japanese/service/products/reagent_plasmid.html .... And it oly costs 220※.
( Just googled the plasmid name pXen05 )
( Just googled the plasmid name pXen05 )
Mega [Andreas Stuermer]
Oct 8, 2013, 4:13:44 AM10/8/13
to diy...@googlegroups.com
sry, its japanese :D
Patrik D'haeseleer
Oct 8, 2013, 4:15:30 AM10/8/13
to diy...@googlegroups.com
By the way - it looks like you may be able to do away with that pesky unstable, membrane-bound, P450-interacting C4H enzyme altogether! The paper I quoted earlier (Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms) also mentions that PAL from R. rubra acts on tyrosine as a substrate as well (page 716 and Figure 5), in which case your pathway would reduce to:
tyrosine --(PAL)--> p-coumaric acid --(4CL)--> p-coumaroyl-CoA --(STS)--> resveratrol
One enzyme less to worry about!
Patrik
tyrosine --(PAL)--> p-coumaric acid --(4CL)--> p-coumaroyl-CoA --(STS)--> resveratrol
One enzyme less to worry about!
Patrik
On Monday, October 7, 2013 7:50:54 PM UTC-7, Patrik D'haeseleer wrote:
Very cool project! I'd love it if you guys could come give a talk about your work at Counter Culture Labs - your friendly neighborhood DIYbio group. You're right in our backyard after all! This is just the kind of projects we'd love to get into once we get our lab up and running.
Do you foresee any trouble synthesizing the p-coumaric acid in lacto? The high-throughput E. coli resveratrol biosynthesis papers I've seen (e.g.
High-Yield Resveratrol Production in Engineered Escherichia coli - 2.3 g/liter resveratrol!) all seem to feed in p-coumaric acid, because getting the C4H enzyme to work in bacteria seems problematic:One of the barriers to the production of flavonoids and their related compounds in microorganisms by means of assembling biosynthetic genes to form an artificial pathway is the difficulty in expression of active, membrane-bound C4H. This enzyme is not expressed efficiently in bacteria due to its instability and the lack of its cognate cytochrome P450 reductase in the host.
Nathan McCorkle
Oct 8, 2013, 4:17:20 AM10/8/13
to diybio
On Tue, Oct 8, 2013 at 1:15 AM, Patrik D'haeseleer <pat...@gmail.com> wrote:
> By the way - it looks like you may be able to do away with that pesky
> unstable, membrane-bound, P450-interacting C4H enzyme altogether! The paper
> I quoted earlier (Combinatorial Biosynthesis of Non-bacterial and Unnatural
> Flavonoids, Stilbenoids and Curcuminoids by Microorganisms) also mentions
> that PAL from R. rubra
...
> By the way - it looks like you may be able to do away with that pesky
> unstable, membrane-bound, P450-interacting C4H enzyme altogether! The paper
> I quoted earlier (Combinatorial Biosynthesis of Non-bacterial and Unnatural
> Flavonoids, Stilbenoids and Curcuminoids by Microorganisms) also mentions
> that PAL from R. rubra
except they want to use only l.spp.bulgaricus and grape genes to calm
the masses.
Nathan McCorkle
Oct 8, 2013, 4:20:04 AM10/8/13
to diybio
On Tue, Oct 8, 2013 at 12:33 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
> Carolina.com sells e.coli ampicillin and a plasmid for $24, presumably plus
> shipping.
Sorry, $24.95 (again pretty sure not including shipping):
> Carolina.com sells e.coli ampicillin and a plasmid for $24, presumably plus
> shipping.
http://www.carolina.com/transformation-dna-transfer-kits/glow-in-the-dark-transformation-4--and-8-station-kit-perishables-refill/211088C.pr?catId=&mCat=&sCat=&ssCat=&trail=SRCH:refill:7002:10146&question=refill
-Nathan
Richard Yu
Oct 8, 2013, 9:29:56 PM10/8/13
to diybio
Exactly Nathan. Though we can probably dig for a food-grade version of a TAL, or perhaps even mutate the grape PAL or HAL to get to mostly TAL function as an intermediate stage to not rouse the pitchforks.
Rich
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Nathan McCorkle
Oct 8, 2013, 10:54:17 PM10/8/13
to diybio
On Mon, Oct 7, 2013 at 8:40 PM, Richard Yu <r...@radiantgenomics.com> wrote:
> Hi Patrick-
>
> Thanks so much for your support!
> Good points. I don't know how Lacto will differ from Ecoli in terms of
> expresion of active C4H, or what the flux will be like in Lacto. We're
> hoping to stick with the simple things- expression levels, perhaps protein
> fusions might also do the trick (analogous to (Zhang, Y et al, JACS
> 128:13030-13031 (2006))). Fusing PAL to C4H, or C4h to 4CL? Get that
> substrate channeling thing going. The farther we get away from native
> proteins, though, the farther we get away from that food-grade criteria for
> a food product. It's an interesting set of scientific and non-scientific
> constraints to match :). Will be fun to see how far we can bring standard
> metabolic engineering and strain improvement techniques into the fold, too.
> (mutagenesis and selection, for example- not GMO!).
Realistically though that logic doesn't make sense. Is it possible to
> Hi Patrick-
>
> Thanks so much for your support!
> Good points. I don't know how Lacto will differ from Ecoli in terms of
> expresion of active C4H, or what the flux will be like in Lacto. We're
> hoping to stick with the simple things- expression levels, perhaps protein
> fusions might also do the trick (analogous to (Zhang, Y et al, JACS
> 128:13030-13031 (2006))). Fusing PAL to C4H, or C4h to 4CL? Get that
> substrate channeling thing going. The farther we get away from native
> proteins, though, the farther we get away from that food-grade criteria for
> a food product. It's an interesting set of scientific and non-scientific
> constraints to match :). Will be fun to see how far we can bring standard
> metabolic engineering and strain improvement techniques into the fold, too.
> (mutagenesis and selection, for example- not GMO!).
not play that card in the marketing department? It just allows
ignorant unscientific claims to claim traction via your teams hard
work and sweat. Please, whatever you do, don't ever allow people to
think GMO is any worse or better than mutational or selective
breeding.
Microarray analyses reveal that plant mutagenesis may induce more
transcriptomic changes than transgene insertion
"Finally, we believe that safety assessment of improved plant
varieties should be carried out on a case-by-case basis and not simply
restricted to foods obtained through genetic engineering."
http://www.pnas.org/content/105/9/3640.full
Nathan McCorkle
Oct 9, 2013, 1:27:46 AM10/9/13
to diybio
I may have been a bit misleading in the last email, as I was ranting more about mutagenesis as it refers to 'shotgun genetic engineering' aka using chemo and radio mutagenesis. With Synthetic Biology *wink wink*, one could simply use a computer program to mutate nucleotide by nucleotide the protein's gene. After a while it might just happen to look like a heterologous gene, but assuredly it wouldn't be cross-species, simply evolved in-silico.
Cathal Garvey
Oct 9, 2013, 6:32:30 AM10/9/13
to diy...@googlegroups.com
Wouldn't error-prone PCR and re-transformation count as "mutagenesis",
without the hazard of genome wide mutation?
Broadly in agreement with you Nathan, that if you're going to do GMO,
just do GMO. Detractors *don't give a crap* how much effort you've put
in, they'll just shout "frankengrapeyoghurt" as loud as they can and
fund thugs to break into your lab and boil your fermenters.
When asked by the media, you tell the truth; "we're using grape genes".
You're going to have to adapt the genes to work in L.bulgaricus anyway;
codon usage, shine-dalgarno, bacterial promoters, potentially a
rho-independent terminator or two. By that time, why does it matter if
you've optimised the gene any further? It's already a
frankengrapeyoghurt by then. Hiding behind appeals to nature and
essentialism just make it seem like there's something wrong with technology.
However, where I disagree with Nathan is when you talk to slightly more
savvy but no less fearmongering regulators. Yes, they may want the genes
to appear as "natural" as suits their essentialist-aesthetic, and so
perhaps the less you change things the more warm they'll be to licensing
it. For one thing, changes to DNA are (AFAIK) never allergenic, but
changes to proteins might be..but they probably have procedures in place
to assess allergenicity potential.
So why not ask them and work so as to avoid the issue? If they just run
a pBLAST against known allergens, you can do that too and reject mutants
that get scores above a certain threshold. Do the regulatory screening
for them, in advance, and make their job easier while not having to tie
your hands back just to appease serfdom-romanticising hippies. :)
without the hazard of genome wide mutation?
Broadly in agreement with you Nathan, that if you're going to do GMO,
just do GMO. Detractors *don't give a crap* how much effort you've put
in, they'll just shout "frankengrapeyoghurt" as loud as they can and
fund thugs to break into your lab and boil your fermenters.
When asked by the media, you tell the truth; "we're using grape genes".
You're going to have to adapt the genes to work in L.bulgaricus anyway;
codon usage, shine-dalgarno, bacterial promoters, potentially a
rho-independent terminator or two. By that time, why does it matter if
you've optimised the gene any further? It's already a
frankengrapeyoghurt by then. Hiding behind appeals to nature and
essentialism just make it seem like there's something wrong with technology.
However, where I disagree with Nathan is when you talk to slightly more
savvy but no less fearmongering regulators. Yes, they may want the genes
to appear as "natural" as suits their essentialist-aesthetic, and so
perhaps the less you change things the more warm they'll be to licensing
it. For one thing, changes to DNA are (AFAIK) never allergenic, but
changes to proteins might be..but they probably have procedures in place
to assess allergenicity potential.
So why not ask them and work so as to avoid the issue? If they just run
a pBLAST against known allergens, you can do that too and reject mutants
that get scores above a certain threshold. Do the regulatory screening
for them, in advance, and make their job easier while not having to tie
your hands back just to appease serfdom-romanticising hippies. :)
Mega [Andreas Stuermer]
Oct 9, 2013, 7:06:01 AM10/9/13
to diy...@googlegroups.com
>Wouldn't error-prone PCR and re->transformation count as "mutagenesis",
Next time you can synthesize it witha computer and it is synbio.
>without the hazard of genome wide mutation?
The first time you do this, yes.
Next time you can synthesize it witha computer and it is synbio.
Mega [Andreas Stuermer]
Oct 9, 2013, 7:07:57 AM10/9/13
to diy...@googlegroups.com
Btw, those who want radical life-extension will like to hear "gmo".
Cathal Garvey (Phone)
Oct 9, 2013, 7:49:45 AM10/9/13
to diy...@googlegroups.com
Chemical/polymerase mutagenesis is far, far cheaper to do than synthesising millions of mutants. The challenge is creating a screening system to pick the "good mutants" from a pool of millions.
Mega [Andreas Stuermer]
Oct 9, 2013, 10:28:46 AM10/9/13
to diy...@googlegroups.com
Some good points were listed... haters keep hating, for sure.
But if you use romanticistic language, also people that were open-minded perheps get sceptical. If you allow conspiracy theories to grow.
But if you use romanticistic language, also people that were open-minded perheps get sceptical. If you allow conspiracy theories to grow.
It's a great product, and there will be people consuming it for sure!
Richard Yu
Oct 9, 2013, 2:30:21 PM10/9/13
to diybio
Thanks all for the stimulating discussion and encouragement!
We're hoping to use native Lacto (or maybe S. thermophillus) parts/DNA as much as possible. Native lacto promoters should be fine for the first pass, both from a scientific point of view and to make it easy for regulators/critics. No sense in making it harder on all of us.
Of the many anti-GMO concerns, one that is legitimate to me is the allergen issue of heterologous proteins. So we have to test allergenicity, just like for of any new food. (I don't know if computational prediction is sufficient; maybe to get the ball rolling, or as a necessary pre-screen). That will also cover any unintended side effects of the engineering (increased expression of something allergenic, change in post-translational mods of native proteins that may increase allergenicity, etc.) Low prob, but might as well check.
We're not trying to lead the pro-GMO charge explicitly here. We _are_ trying to be clear about exactly what we're doing without needlessly provoking anyone. I think releasing a good, safe engineered product is one of the best way to provide good press to the whole movement.
Best,
Rich
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Mega [Andreas Stuermer]
Oct 9, 2013, 4:17:25 PM10/9/13
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I think releasing a good, safe engineered product is one of the best way to provide good press to the whole movement.
Exactly!!
I guess many people think "what can GMO do for me?"
Usual GM products like GM crops are beneficial just for the farmers and carry the discussion of herbicide resistance etc.
So, if it's not any more healthy than a "natural" product, 99% will choose the natural.
You are turning this situation around. Great work!
Patrik D'haeseleer
Oct 9, 2013, 6:37:48 PM10/9/13
to diy...@googlegroups.com
Actually, it looks like there's a tyrosine ammonia-lyase in tea, strawberries and corn as well, and phenylalanine/tyrosine ammonia-lyase in soy, sunflower, sorghum and wheat:
http://www.brenda-enzymes.org/php/flat_result.php4?ecno=4.3.1.23&organism_list=&Suchword=&UniProtAcc=#ORGANISM
http://www.brenda-enzymes.org/php/flat_result.php4?ecno=4.3.1.25&organism_list=&Suchword=&UniProtAcc=#ORGANISM
Not sure that the genes for these activities have been annotated in all of them, but there should not be a shortage of TAL sequences from food plants to play with.
Hm... BRENDA actually also lists PAL4a and PAL5a from Vitis amurensis as being tyrosine ammonia-lyases. Haven't tracked down where that annotation comes from, bu if true, you might be able to do your design purely based on (different species of) grape.
Patrik
http://www.brenda-enzymes.org/php/flat_result.php4?ecno=4.3.1.23&organism_list=&Suchword=&UniProtAcc=#ORGANISM
http://www.brenda-enzymes.org/php/flat_result.php4?ecno=4.3.1.25&organism_list=&Suchword=&UniProtAcc=#ORGANISM
Not sure that the genes for these activities have been annotated in all of them, but there should not be a shortage of TAL sequences from food plants to play with.
Hm... BRENDA actually also lists PAL4a and PAL5a from Vitis amurensis as being tyrosine ammonia-lyases. Haven't tracked down where that annotation comes from, bu if true, you might be able to do your design purely based on (different species of) grape.
Patrik
Richard Yu
Oct 9, 2013, 7:10:04 PM10/9/13
to diybio
Cool Patrik! I'm glad my assumption of genetically encoded TAL activity elsewhere in the edible world was a good one. Defiinitely some other enzymes to try... if we can this thing off the ground!
-R
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Mega [Andreas Stuermer]
Oct 10, 2013, 2:29:13 AM10/10/13
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Found out: 1700 euros for the jabanese gram positive lux transposon....
Patrik D'haeseleer
Oct 10, 2013, 7:26:07 PM10/10/13
to diy...@googlegroups.com
On Wednesday, October 9, 2013 11:29:13 PM UTC-7, Mega [Andreas Stuermer] wrote:
Found out: 1700 euros for the jabanese gram positive lux transposon....
Luckily, only one of us would have to pay that. ;-)
Hm... perhaps it might be time to do a little crowdfunding. I've seen CrowdTilt used as a very light-weight crowdfunding platform to pool funds from a group of people.
Patrik
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