 Exploitation of animals on the industrial scale that we practice it now is going to end is going to 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 in a manufacturer that happened 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 hacker space actually two hacker spaces bio curious and sunny veil and counterculture labs in Oakland We are engineering bakers east also the same use yeast used to make beer To produce casein the protein and 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 hacker spaces 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 I've 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 hacker spaces 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 of 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 we're looking just not at dairy it's actually over half of the water used in the United States and There's more greenhouse gas emissions worldwide from animal agriculture than there is from transportation So 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 bunch of cows on dry destroyed land This actually comes from a big farm just north of LA that a lot of people from California have probably seen the other one is a 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 Counterculture labs is in Oakland. That's where I'm doing my part of the work And then bio curious 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 gonna 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 that's three of the casein proteins casein is 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 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 us 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. I've a couple 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 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 did 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 know that though that you can pull on it and 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 you know 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 of 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 a 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 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 In 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 an mammal we are not actually doing this ourselves other people are doing this university Has 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 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 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 isms 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, that's oh so Yeast glycosylate proteins where bacteria do not so E. Coli is used for we're making some things that you may have heard of genetic engineering 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 a little bit so We're putting this information into yeast. I told you it's going to be a DNA, but you know 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 gonna put in It's 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 with this 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 itself on its own would not really work So this is a this is from the Wikipedia page. So it's showing bacterial not yeast but same idea That plasmid right now the way we're doing it does not get inserted into the Main part of the organisms 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 gonna 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 gonna make each protein separately and then recombine them later This is much easier to optimize one protein and each yeast and not try to make all four and 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'll 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 gonna have to play around. We've not scaled up large enough to have large amounts of the protein yet We're gonna 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 You know a lot of people are against GMOs, you know, there's protests against it And the US at least saying GMO free is now a selling point for a lot of things And that's 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 or evil GMOs in Monsanto or one the same thing And the biggest Actual problem is Monsanto has sued farmers for saving seeds and replanting them the next year, you know 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 Milkweeds being killed off and Monarch butterflies are endangered. So this is one concrete example of a bad side effect of this GMO crop but 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 have come out genetic engineering And there's actually a lot more of these in those couple of bad examples So chymison, this is the enzyme from rennet. This is what's used in cheese making It used to be and the small amount so 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 chymison to make all that cheese Also, the activity was really variable. It's coming out for this living being You know, we're all different the The rent it was not of predictable quality So now 80% of the rent 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 the coal. I 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 we can change the insulin a little bit and make it either faster acting or longer ass 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 what Yeah figure out the 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 in our wall because you know why not This this was my favorite quote real vegan narwhal cheese while I'm sure synthetic biology can get weirder But this is a good start That was from That was from a Riley radar Okay, um, yeah, I think of that time So I'm not gonna go through all these bullet bullet points one by one But we've built a functional organization. That's not hierarchical. It's not always easy to do as some of you probably know Um, 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 that 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 in a 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 have we're getting protein out that is the right size to be the protein 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 It's probably what you'd expect at least from my point of view so 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 gonna 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 case in so And 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 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 is 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 it's also an outreach Project you know 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 just I'll just say that at first actually will 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 Here we go And if so even at 2.5 grams per liter We get one half the 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 go vegan. I hope so but 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 three hundred million dollar buyout offer from Google because they said that was way too low They're trying to disrupt a one point three trillion dollar Industry so they think three hundred 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 do 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 is on the bottom there and I'm staying at nope the Bay Area Hacker space 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, and I wanted to ask you is there any project about the flavor the flavor of the things meaning I think that they are used very much in the in the industry and they can be some sort of scaling up a mechanism for all these Is there any project about that for flavorings? You mean not specifically for cheese or you mean specifically for cheese Bacon cheese a cheese man that tastes like bacon, you know, 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 Is happening now one more go for it I was just wondering is there a way we can follow this project like 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 like simply and easily without necessarily contributing to it? Yeah, I mean Our mailing list would be one place, but maybe not the best if you just want to get like highlights of what we've accomplished We do have a Facebook page that Unfortunately, I'm responsible for and don't update enough We also we do have a wiki that does have like lab notes and And is pretty up-to-date so that would be the best place if you just go to a real vegan cheese org It's got all of our contact information and the wiki even find them there Okay, that's one Hello, my name is your your yana. I'm from a Biotincra space here in Berlin and I have a question How long does it take that you got your lap and to run? And how long does it take that you got your project started right so Bio curious 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 of 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 it is if you go in the space. It does not look very built out We just have the equipment we have and we have bench space. You know, 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 We 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 breaking 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 Thank you