exploitation of animals on the industrial scale that we practice it now is going to end.

This will happen within most of our lifetimes, and activists will deserve some credit for it, but really it's not because we will decide to become better people collectively, it's because people will make better products, things that taste better, perform better,

but also are cheaper and use less resources to manufacture that happen to be vegan, and that's how it's going to happen. I'm going to talk about a project along these lines today. It's called Real Vegan Cheese. So what is this? What we're doing at a hackerspace, actually two hackerspaces,

Biocurious and Sunnyvale and Countercultural Labs in Oakland, we are engineering baker's yeast, also the same yeast used to make beer, to produce casein, the protein in milk that gives milk and cheese its most important properties. So this is a genetic engineering project.

So there's a really wide group of people doing this, being hackerspaces, and we've got everything from PhD biologists who have a lot of experience with this sort of thing, to high school students, a lot of people with maybe computer engineering backgrounds,

computer security that have never done any biology before. I'm a chemist, not a biologist, so we've got a really wide group of people. And so we also have a wide variety of reasons for wanting to do this. So I led with the animal rights reason. Not everybody is so into that in the group.

There's only a couple of vegans in the group, most of us are not. So the project started actually as a way, several things, to show that hackerspaces can compete with universities and large corporations for doing relevant work, interesting work. Also to give people a pathway to learn synthetic biology,

without going to grad school, just coming into the lab and learning to do it. I have the animal rights reason, but then there's also the environmental reasons apart from the animal rights. And I'll get to that in a second. But from my perspective, what this project is,

is not a way to make better vegan cheese for vegans, but a way to make cheese which is vegan that everybody else will accept. So the environmental reasons though are the ones that pretty much the entire group agrees on. So I'll talk about that a little bit.

There are 270 million dairy cows in the world. That's quite a few cows, that's close to the human population in the United States. And so this causes a lot of environmental impact. They're responsible for about 4% of the greenhouse gas emissions in the world. Uses a lot of water, more than almond milk, which you may have heard of.

Also there's a lot of pollutants run off into waterways. And this was an interesting graph that I found in one academic paper I read. And I'm not sure how clearly you can see it, but what it shows, the two biggest bars that show the amount of CO2 emitted are from red meat,

and then the next one is from dairy. If you look at the smaller bars in the graphs, it basically says we should all be eating in restaurants, ordering beverages, and eating miscellaneous other, which is great because that's pretty much my diet.

So the broader impact of animal agriculture, if you're looking just not at dairy, it's actually over half of the water use in the United States, and there's more greenhouse gas emissions worldwide from animal agriculture than there is from transportation. And it uses a third of our ice-free land.

Visually you can see just how intensive this is, just looking at the farms. The one picture is that's a bunch of cows on dry, destroyed land. This actually comes from a big farm just north of L.A. that a lot of people from California have probably seen. The other one is an aerial view of a manure runoff pit.

That big red thing is a manure pit, and you probably can't see it, but there's a bunch of little black dots north of that pit or above it, and those are individual cows, so that manure pit is huge. Just quickly, I said we're two different labs.

Counter Culture Labs is in Oakland. That's where I'm doing my part of the work. And then Biocurious is one of the earlier biohacker spaces, and that's in Sunnyvale. So we're both around the Bay Area. Okay, so now I'm going to shift a little bit

to some of the technical part of the work itself. And so why are we engineering casein, and what's so special about this? Well, if you look at the kind of ball on its own, the yellow and blue ball on one side, the blue stuff inside, that's three of the casein proteins.

Casein's actually four different proteins. Three of them are really hydrophobic. They don't like interacting with water, so they all ball together. Then the fourth one, kappa casein. This is really important. It's got a hydrophobic side, so it wants to be near those other hydrophobic proteins. It's also got a hydrophilic side. It likes to be in the water, so it forms this shell around and solubilizes the whole thing.

It means that you can get it into suspension. And there's also a bunch of calcium on the inside with that hydrophobic part that's attached to the protein by phosphate groups. It's not really important to know what a phosphate group is, but it's not something that is on the protein when it's expressed. It's something that has to be added later.

And this is important. The hydrophilic part, the part of the kappa casein that holds this all in water, it's also important, it has a bunch of sugars on it. These sugars also are not there when the protein is initially expressed. It's added later. These steps are called post-translational modification, and it's something that's hugely important to the functions of proteins,

so we can't ignore it. But what about just... Okay, so this casein... Oh, sorry. A couple of things I forgot there, important. So when you're making cheese, what happens is you cleave off this hydrophilic part of the outside of the protein. So it's no longer soluble in water.

So all these globules of protein and calcium stick together. Some of the water separates out, and as you do this more and more, you get this three-dimensional network. That is what cheese is, pretty much. It's this three-dimensional network of bonded, but not very strongly bonded globules.

So the not very strongly bonded is important, because when you heat it up, you overcome that bonding energy and it melts. That's why cheese melts. It sticks together well enough, though, that you can pull on it and it stretches and will spring back to some extent, but if you pull too hard, it will rip. So this is what gives cheese its properties.

So what if we just want to reproduce this? People have been trying to do this for a while. You can buy vegan cheeses of various qualities now. Some of them do melt. Unfortunately, the ones that melt are not the ones that taste okay, for the most part, in my opinion. So what's this, you know, why do mammals make casein,

but we don't have anything else like this? Well, casein is really specifically made for young mammals. They need a lot of specific nutrients, and this was an evolutionary solution to that, where we can pack in all this protein, we can get more fat in, get more calcium in,

then you could dissolve in water and most other ways you could think of. So this was a very clever solution. So as far as we know, there's nothing in the plant kingdom and yeast now, anything like this, that is similar enough to use it. So there's some chance that someone will come up with another way to do this, but really,

if we want casein or something very similar, we need to genetically modify an organism to actually produce that. That's going to be the best way to give us the properties we want. So what do we actually do here? So the first thing we do is we sequence the genome of a mammal.

We are not actually doing this ourselves, other people are doing this, universities have been doing it for a long time. So we then search those sequences for the parts that express the milk proteins. So we find that piece of DNA, we then, we just pull that information from a database, and then we get a commercial lab to synthesize that piece of DNA

and some other information that we need to include with it. And so we get the DNA made. We then insert that gene into yeast. So now the yeast should start expressing it if we've done all the work correctly up to this point. Once it starts expressing, we have to isolate and purify it.

This is also important, and this can be the most difficult step in many cases. Once we have that, we need to formulate a milk. We need to add sugars and fats, and we're not trying to reproduce the exact sugars and fats from milk or cheese, we're trying to reproduce the protein,

and then other sugars and fats are close enough that we should be able to find things that are already vegan that we can just use. So you can use other sugars, other fats. Once we have that, we can make cheese, and we can make cheese exactly in the same way that animal cheese is made now. The same cultures, same enzymes.

Okay, so I told you we're doing this in yeast, but why choose yeast? There's a lot of different microorganisms out there. There's plants, other things. Well, if you look on the tree of life, up here in the eukaryotes, animals and fungi are actually very close together.

On the cell level, a lot of our machinery works in the same way, or similar enough that we can engineer these proteins into the yeast and expect some of the same post-translational modifications to occur, which is important to us. Okay, so yeast glycosylate proteins,

where bacteria do not, so E. coli is used for making some things that you may have heard of genetic engineering of E. coli, but they do not glycosylate things. These are the sugars that go on the surface that make the whole micelle more soluble. So we're hoping that the native glycosylation will be enough.

We haven't actually gotten quite this far, and I'll tell you more about the progress in a little bit. So we're putting this information into yeast. I told you it's going to be a DNA, but exactly how do you do that? You know, it's not quite that easy, but it's actually not all that hard.

So the DNA we're going to put in is called a plasmid. It's got the sequence to express the protein we want, but that sequence is not the same as the DNA sequence in the mammal. It's actually been optimized for yeast,

because when yeast reads this DNA, it will act slightly differently given the same base pairs. So we actually modify the base pairs to encode optimally for yeast. We then put some other information in there. We need some other functions. I'm not really going to talk about that, but that piece of information on its own would not really work.

This is from the Wikipedia page, so it's showing bacterial, not yeast, but it's the same idea. That plasmid right now, the way we're doing it, does not get inserted into the main part of the organism's DNA. It actually exists as a separate piece of DNA,

but it is still expressed. So once we've got this DNA into the yeast, we need to actually make our proteins and isolate them. So we're going to do this in bioreactors. They're just big, stirred glass reactors. Our main input will be sugar.

Not actually table sugar, but it's a good visual aid. And I told you there are four different proteins. Well, we're actually going to make each protein separately and then recombine them later. This is much easier to optimize one protein in each yeast

and not try to make all four in a single yeast cell. It also turns out that you don't really need all four. You can make these micelles with just two of these, so we may not have to do the whole thing. That will make our work a lot easier. In that plasmid, some of the other information I said we have, there's a transport tag.

So as the yeast is making this protein, there's actually a tag on it that says kick it out of the cell, and as you're doing it, cut that tag off. This is a really convenient thing to have because we can get the protein outside of the yeast. And so we'll have this collection of yeast and protein, the yellow and blue dots. We can filter it, and then we'll have a solution

that's mostly just the protein. Unfortunately, this is probably not quite enough, and we're going to have to play around. We've not scaled up large enough to have large amounts of the protein yet. We're going to have to play around with some probably chromatography or hopefully just changing the pH to crash out the protein.

So once we have these purified, we combine two to four of them, and it reconstructs into the micelle. Now, we actually know this works already. How do we know it works? Well, we're making identical proteins to what cows produce, and so these proteins have already been pulled apart before

and put back together, so we already know that that step can work. Once we do that, we can formulate it with the sugars and fats, and then we have our milk to make the cheese. So now I want to talk a little bit about some of the philosophical issues. A lot of people are against GMOs. There's protests against it. In the U.S. at least, saying GMO-free

is now a selling point for a lot of things, and besides all the other things I've talked about, that's part of my group's mission is to also change the perception of GMO. It's not to say that there's no problems at all, but this is the image most people have, is that GMOs are evil.

GMOs and Monsanto are one and the same thing. And the biggest actual problem is Monsanto has sued farmers for saving seeds and replanting them next year. But this has been the practice for millennia. This is how agriculture has worked up until very recently.

So the problem with seeing that as an inherent evil in GMO is that conventional crops can also be patented. The European Patent Office actually just upheld several patents on broccoli and tomatoes. So really, the problem is patents and corporate behavior, not genetic modification.

There's also another actual problem. Roundup-ready or glyphosate-ready crops. These are not maybe the problem that most people think they are, but what the real problem is is that when Roundup is sprayed on crops,

all the other plants die. And not just in the field, it's often over-sprayed and things around the field die. So one concrete example of how this is a problem is in the United States, milkweed and butterflies. Monarch butterflies only survive on milkweed. Milkweed mostly grows in areas where corn and soybeans are grown.

Milkweed is being killed off and monarch butterflies are endangered. So this is one concrete example of a bad side effect of this GMO crop. And it can be dangerous to the people spraying the Roundup, but if done according to specs, it's actually not that dangerous. It is actually safe for us to eat. By the time it gets to us, it's okay.

And despite some claims to the contrary, most of the literature on the subject agrees that crop yields actually are increased. On the other hand, there are some really good things that have come out of genetic engineering, and there's actually a lot more of these than those couple of bad examples.

So chymosin, this is the enzyme from rennet. This is what's used in cheese-making. It used to be, and a small amount still is, it used to be extracted from the stomachs of slaughtered calves, but there's a few problems with this. We didn't have enough supply. People eat a lot of cheese. Even with all the calves we slaughter in the dairy industry,

there was not enough chymosin to make all that cheese. Also, the activity was really variable. It's coming out of this living being. We're all different. The rennet was not of predictable quality. So now 80% of the rennet in the world is made in E. coli and bioreactors. So if you eat cheese,

you're eating the product of a genetically engineered organism already. So our vegan cheese will not really be all that different philosophically. A bigger deal is insulin. We used to extract insulin from the pancreas of pigs.

This was a huge problem. This was even a bigger supply problem. We didn't have nearly enough of it. A lot of diabetics were untreated. Again, it was very variable in quality, and some people were actually being poisoned by some of the byproducts, having immune reactions and such. So now we again make it in... I believe this one's in E. coli.

We make it in a genetically engineered organism. We have an abundant supply. The high prices are all just corporate behavior. It's not fundamentally because it has to be high. It's very reliable, and the other great thing that's happened is we figured out how to modify it. We can change the insulin a little bit and make it either faster-acting or longer-lasting.

These are not things we could do easily from something we've extracted from a pig. So I mentioned before that we searched the genome of different mammals to figure out where the milk-making genes are,

and so we're doing cow, which everybody expects. We want to do human, and this kind of grosses some people out at first, gets a kind of strange reaction, but really it is the one that should have the lowest immune response. People can be allergic to cow proteins,

and our engineered proteins should be no different. So the human proteins should be the ones that we have the least chance of having a reaction to, because everybody already makes these, males included, at least in small amounts. We're also going to do narwhal, because, you know, why not?

This was my favorite quote. Real vegan narwhal cheese? Well, I'm sure synthetic biology can get weirder, but this is a good start. That was from... That was from O'Reilly Radar. Okay, yeah, I think I've got time.

So I'm not going to go through all these bullet points one by one, but we've built a functional organization that's non-hierarchical. It's not always easy to do, as some of you probably know. We've been talking to the FDA. They're freaked out about the human protein, because they're afraid of autoimmune responses,

sort of exactly the opposite of why we wanted to do it. They're perfectly fine with a cow, so we'll probably get approval relatively quickly. They didn't really have a reaction to the narwhal. Something some of the members of the group have been doing, I've mentioned that we're not all vegan and that we have many different motivations.

Some of us are actually taking casein that's isolated from cow's milk and trying to reconstruct milk and then cheese using our other vegan ingredients, so when we have enough of the protein, we're ready to go with the rest of the steps. We know it all works. So in the lab, we've made a bunch of new plasmids.

The first thing you do with them is that you put them into E. coli and you get them into replicate so that you have enough to then put into your yeast. So that's worked in 10 of the 11 steps. One of them actually turned out to be toxic to the E. coli itself. This happens sometimes, but it turns out that was one of the least important for us to have,

so that's fine. So we've confirmed that our plasmids work. We've put it into a yeast. We had some difficulties, as is normal for lab work. But we're getting protein out that is the right size to be the proteins we expect, and so this is where we are right now.

We believe we're getting some of the correct proteins back out. Now, we've been working on this for a while. It may seem like slow progress, but consider that we're only a few biologists and we're a completely volunteer organization working like one or two nights a week. It's actually pretty good. For the amount of lab time we put in,

it's probably what you'd expect, at least from my point of view. So we're able to make some proteins, but we've not actually made cheese yet. Something else we did is we went to the iGEM competition, and I'm going to just start out showing the trophy and then I'll explain what this is. So the iGEM competition is a worldwide competition.

It's the International Genetically Engineered Machines Competition. So this was originally an undergrad competition, but they opened it up last year to community labs also. So we won best community lab and overall got a really good response from the community.

Okay, so obviously we have a lot more to do just even on the bovine casein. So the biggest two things, actually, are figuring out if we need to do further modification, the post-translational modification.

We don't actually know this part yet. We think we're making some of the right proteins. We don't know if they're going to work exactly as how we expect. And the purification. These are non-trivial. This is a long project, and we're probably closer to the beginning than the end of it. At that point, we actually have funding to buy a bioreactor. We have people that know how to use them,

so we'll be able to scale up once we've proven for sure that we have exactly what we want. And then we move on to Narwhal and repeat the whole process. And so when we go to the bioreactor, we're going to work on a 50-liter scale at first. Unfortunately, our first target's only 2.5 grams per liter of protein,

so this is not going to be a lot at first. But we think we can optimize beyond that. Part of this whole thing, as I mentioned, is also an outreach project, talking to people about how GMOs are not actually evil, how we can replace animal products with vegan products that are just as good.

And so we've actually got quite a bit of press. And I already went over some of this, and I'm running out of time. So I'll just say that at first, actually, we'll probably be in the specially vegan product price range, but we believe that we can bring that down.

We'll have to beat that 2.5 grams per liter, but we can bring that down, I believe. There we go. And if so, even at 2.5 grams per liter, we get one-half the greenhouse gas output for the given amount of cheese. So we've already made a big improvement.

It's lower water usage. Even if we never get to that price, which I think we will, we may encourage more people to go vegan. I hope so. The other thing is, this is all open source. You can find all of our work online. So you can reproduce any of this yourself. If you feel like it, you can improve on it.

We are competing with some commercial operations, trying to get to patents first, so to make this open for anybody. And some of our team members have this vision of being able to make vegan cheese in your own kitchen, just have a yeast culture that's paying out protein and that you can go and culture to cheese. This would be very inexpensive. You'd only have to pay for sugar and water, basically.

Maybe a few other things. I'll skip over this. Just say there's a lot of commercial companies out there trying to do the same thing for all kinds of animal-derived products. Some of them are doing very well. In fact, Impossible Foods just turned down a $300 million buyout offer from Google,

because they said that was way too low. They're trying to disrupt a $1.3 trillion industry, so they think $300 million is too small. So if any of you want to get involved, if you're in the Bay Area, you can come by one of the labs. You can also remotely, if you want to help out with some organizational work.

But the other thing I would encourage you to do is to just go start your own project along these lines or something similar. We'd be happy to talk to you about it if you want to try to actually do the vegan cheese. Specifically, we can share our plasmids. We can share what we've done so far, all the details. And you can also get in contact with me,

and my information's on the bottom there, and I'm staying at NOPE, the Bay Area hackerspace camp that's right over here. So, yeah, I think go ahead and take some questions.

Thank you. Now we have a couple of minutes for some questions. If anyone would like to ask a pressing question, please step on down to the microphone. We've just got a couple of minutes, so run on down. Hello. I wanted to ask you, is there any project about the flavor of the things?

Meaning, I think that they are used very much in the industry, and they can be some sort of scaling up mechanism for all these. Is there any project about that? For flavorings? You mean not specifically for cheese?

Not specifically for cheese. For example, bacon cheese that tastes like bacon. Yeah, there are a lot of efforts along these lines. Vanilla is now made. There's a lot of natural vanilla still. Some of it is actually from wood pulp. You can get vanilla out of the byproducts of wood pulp, if you process it correctly, but there's also genetically engineered organism, vanilla.

And I'm sure lots of other flavorings, but that's the one that's actually happening now. One more. Go for it. I was just wondering, is there a way we can follow this project? I live in Australia, so I can't come and pop by your lab,

but is there a way we can follow this project very simply and easily without necessarily contributing to it? Our mailing list would be one place, but maybe not the best if you just want to get highlights of what we've accomplished. We do have a Facebook page that, unfortunately,

I'm responsible for and don't update enough. We do have a wiki that does have lab notes and is pretty up-to-date, so that would be the best place. If you just go to realvegancheese.org, it's got all of our contact information and the wiki. You can find them there.

Thank you. Okay, last one. Hello, my name is Joanna. I'm from a biotinkler space here in Berlin, and I have a question. How long does it take that you got your lab to run, and how long does it take that you got your project started?

Biocurious has been around for five or six years at least. I don't remember when it started. They were up and running, had a bunch of equipment before this project started about a year ago. Counterculture Labs, actually, when I started, was a small room off someone's kitchen in Oakland,

and actually, that's all you really need, but we do have a lab space now. It took us a few months to find that space, but to build it out, it took us, it's been at least six months, and really, if you go in the space, it does not look very built out. We just have the equipment, and we have bench space. So it's taken a while.

The project, I mean, we made a very quick initial project. I was not there for the first couple months before the lab work started, but then once I joined, we got a lot of our initial results within a month or so, even not doing a lot of lab work, but we also didn't have funding at the time,

so we ran an Indiegogo campaign last summer, which was very successful. We got, which I'm forgetting now, I think like $38,000, so that's what's been sustaining us now. Okay, thank you. So the future of cheese is rather tasty. Oh, sorry, it was really bad too.

Thank you very much. Another warm... Yeah, there we go. Thank you.