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
On Mon, Oct 7, 2013 at 7:50 PM, Patrik D'haeseleer <patrikd@gmail.com> 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.
Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms
Patrik
On Monday, October 7, 2013 3:07:38 PM UTC-7, Richard Yu wrote: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,RichOn Mon, Oct 7, 2013 at 2:44 PM, Josiah Zayner <josiah...@gmail.com> wrote:There is already a patent for this process: http://www.google.com/patents/WO2006124999A3?cl=enThough 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?
On Sunday, October 6, 2013 4:43:27 PM UTC-5, Richard Yu wrote:Hi Andreas,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)[...]--
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