--:-- Hannah Winarsky: Could you reverse engineer these networks? And let's actually explain what you mean by that, reverse engineering a large-scale network, because that's not a concept that's native to traditional. It's not a bio, it's an engineering concept. So it's a strong electrical engineering concept. So the notion is that you have a system that's interconnected or wired together. And so the example that could relate to many listeners would be the wiring system or the wiring setup of your house. Say you buy a new house, and you've got a circuit breaker box down in the basement, and let's say it's not labeled by the previous owners. Just like my house. Just like my house. So what do you do? You go down and you flip the different switches on or off, and you discover what they connect. So you're beginning to reverse engineer the circuitry of your house. You then, through additional experiments, you're running the microwave, same time with your toaster, that in fact, oh, those two circuits are actually interconnected even further that's not represented by that. And you begin to infer the underlying structure of the wiring diagram of your house. But our first response was to run away from that problem as fast as we could because there weren't sufficient data to do it. In the late 90s, we just had microarrays appear, technology that is no longer in vogue, but would allow you to survey thousands of genes and their expression states simultaneously within a given cell. These were incredibly expensive. And when I got pulled in, there were only seven publicly available data sets. So the idea you could reverse engineer a large-scale network was ludicrous. And what were those types of data that were lacking? So there was largely expression data set. And in those days, it was really microarrays. So even though the technology just appeared, we didn't have those data sets. We didn't have a wet lab. We didn't have the capability. And so we began to think about, could you put together molecular parts in a network? Could we instead take a tinkerer's approach to molecular biology? So the expression is the output. What's the input? Like, how were you perturbing it then? Well, so on that side, we had no perturbation, right? Perturbation would be that you're going to kick the system or you're going to stimulate the system in some way. Or flip the light switch. Flip the light switch on. Let's say you have your radio on in your kitchen upstairs with the volume on. You're going to run downstairs. You're going to flip the switches one by one until you hear the volume go off. That's a perturbation to your circuit that allows you to map out the circuit. We introduced techniques to do that. But even before we get there, we stumbled into what became synthetic biology. We said, okay, could we instead of figuring out how these large-scale networks are wired together, could we put them together ourselves? Could we do it with intent? So could we design circuits with the desired architecture and a desired function? And that's the key difference, right, in this entire approach that you're designing. Exactly. We are really doing biology by design. In some cases, we're taking natural parts and putting them in new and different ways. in other cases, we're building new parts and then putting those together in new or even established architectures. We spent about nine months thinking about what we wanted to build, and we arrived at building a genetic toggle switch. And this was motivated by electrical engineering, where toggle switches, which are also called flip-flops, RS latches, are very simple memory elements, memory switches that can be flipped between zero, one in the binary state or on, off by just a transient electrical signal. Toggle switches are at the heart of digital memory. and we cycled through many different schematics and circuit diagrams and arrived at one that was basically a mutually inhibitory network where we have two genes that are set up so they always want to be on, but they're arranged so they're trying to shut each other off. And we showed mathematically and computationally that you should be able to have it as a bistable system. It either wants to exist in state A, gene 1 is on and gene 2 is off, or state B, gene 2 is on and gene 1 is off. And in principle, you should be able to flip it between those states by translating, delivering a chemical or a biomoters that would shut off your currently active gene, allowing the gene that had been kept off by that active gene, allowed to come on that now would produce enough protein that would shut off the gene that had been on. And you can remove your stimulus and you flip from state A to state B or state B to state A. It's like electrical engineering, except not electrons. It's now a wet circuit. But in this case, we actually did mathematical modeling computation. And now the next segue was I reached out to Eric Eisenstadt, who was a program officer at the Office of Native Research who was running a gene circuit modeling program. And I told him what we were doing, and I more or less called him once a month for over about six months and said, could you give us money to try to build this in a lab? And he kept putting me off, putting me off. And I basically wore him down. I said, you know, I think this could be really big, but we need to be able to build this, and I'd like this to be part of your program. And was the reason why it wasn't clearly, I mean, clearly having an on-off switch like that seems incredibly powerful. Why was it not immediately just because it seemed so hard to do? We had talked to about six or seven very serious molecular biologists who told us this couldn't happen, this couldn't work. They said for it to work, you're going to have to have a very tight off state, two very tight off states. That's going to be impossible. You're going to have to introduce a very large plasmid. It's going to put a burden on the cell. The cell's not going to like this foreign element. The joke was they usually pat us on our heads and say, boy, stick to engineering because biology is really complicated. I mean, those do sound like reasonable concerns. I had an incredibly talented graduate student, Tim Gardner, who was a mechanical engineering trained student out of Princeton. Tim presented our modeling. This is a famous ONR meeting where he was more or less shouted off the stage by some very serious molecular biologists, including a future Nobel Prize winner, who said it was impossible. This could not work. And in part Tim who was green in molecular biology had been a mechanical engineer who had designed autonomous helicopters to fly around Princeton didn know any molecular biology presented this engineering concept Nonetheless in nine months he had a functioning bistable toggle switch At the same time we were doing this Michael Illewitz and Stan Liebler who were two physicists sitting in both the physics department at Princeton and the molecular biology department at Princeton, were taking a remarkably similar approach to synthetic gene circuits, completely independent of us and embeknownst to us, in that we worked in E. coli with three different repressor proteins using dynamical modeling. They worked in E. coli with the same three repressive proteins using dynamic modeling. And instead of creating a toggle switch that consisted of two genes trying to shut each other off, they created what they called a repressilator, a ring oscillator, similarly motivated by electronics and electrical engineering. They consisted of three genes in series. A tries to shut off B, B tries to shut off C, C tries to shut off A. Well, it's the same parts, just different circuits. Exactly the same parts, different circuits. They submitted to Nature. We submit ours to Nature. They end up being published at the launch, the turn of the century, in January, late January, in Nature Back to Back. The next week, the head editorial said, oh, you know, physicists are beginning to move into molecular biology. And he said, you should look back at those two papers because there's something interesting going on. You know, you raise an interesting point about how everyone on the biology side thought this wasn't possible. Almost like the biologists, some of them have an immune response against engineering. That's a great way to put it. And that seems to continue even to today. It is, and I have many, many close friends and colleagues who are biologists, but I think within different academic disciplines, there's often too much tribalism. So the very common critique that we get is, oh, they're not biologists, or they're not chemists, or they don't know basic biology, they don't know basic chemistry. And I, yes, yes, yes, yes, and yes. I don't think it's fair, though, and I think that these problems really can benefit from an interdisciplinary approach and different perspectives. So we don't know the detail at the level that the biologists do. And one of the more common critiques we'll get any time we submit papers to molecular biology journals is that all the columns have, they don't have sufficient controls that were done. But I maintain that in almost every biology experiment paper I've read, they've never had the controls that I'd like to see, which is the idea that you can knock out a gene and that what's lacking now can be attributed to the gene is absurd from a system standpoint. It's interesting because, you know, if you think about an electroengineer to a physicist, an electroengineer might not understand like a PNP junction and a transistor the way a physicist might. Right. but can still use them and make parts out of them and still make things. And so I think there is just a role for each one of them. But there's a fundamental difference between sort of doing science and doing engineering. Yes. And that culture clash is pretty distinct. Yeah, I was just going to say it's not entirely just that academic tribalism, right? It's a discomfort with a different kind of mode of knowledge. I would love to hear if you can describe a little bit more what beyond the tribalism is valid, what you try to think about or what you reject entirely. Yeah, no, it's fair. I think the differences in science and engineering are healthy and valuable. I think in many cases, engineering benefits from science in that most of the work that we do from an applied standpoint is really looking to see in what way can we utilize or exploit principles, discoveries that have been made in science and moved toward an application. In the same way, science benefits tremendously from engineering in that as a new technology gets developed, it can just open up the ability to address and answer new and different questions. And so there really is a great synergy, and it becomes interesting on the conference. Well, there's a synergy, but there is still a sort of feminine difference in how they view the world. It is very different, right, in that the engineer often is very purposeful, whereas the scientist is really driven more by curiosity. Can we discover? Can we learn more? And it's not that the engineer doesn't want to learn more. An engineer can do remarkably well in many instances with little, if any, understanding of mechanism. And that we'll get after a black box. You can represent a model that gives you some sense of relation to the input and output of, say, going back to our perturbation. And yet that model may have little, if anything, to do with the underlying biology, but it nonetheless has predictive value that's useful to get after a given application of purpose. That drives my biologist friends nutty. But that doesn't mean that the engineer is uninterested in mechanism. that the more we know about mechanism, the better we can do, the more facile we are. And the more predictive of the model. The more predictive of the model, absolutely. Let's talk quickly about the bacteria. So why E. coli? You know, on E. coli, the E. coli was easy to pick. It was and remains really the workhorse, broadly molecular biology. It's been very well studied. It's still not fully understood, but we had a number of well-characterized parts, established protocols, and so a couple engineers could relatively quickly get up to speed to do experiments on it. Having said all that, I'll give two points. One is that when I first moved into space, I was told not to work on bacteria because everything's been worked out, we understand. And it turned out to be far from true. And second, linked to that is that it's fascinating to me that there still is a high percentage of the genes that are not functioning out of state of Ne. coli, 30 to 40%. Nonetheless, it was enough there that we could begin to design and build with some level of predictability and some level of comfort that you could insert these circuits and get them to function the way you like. Can you tell us about the latest in bacteria that you guys are now working on? There was a very nice piece by Louis Serrano and Attila Becksky that came out also in E. coli about four months later in Nature. And then it was about two and a half years of nothing coming out because the field didn't exist. You had a number of modeling papers, but nobody really had experiments going. We and others then got excited about engineering bacteria, in particular E. coli, as living technologies, as programmable cells. They could sense something in the environment, make a decision, and act on it. And around 2004, began creating these programmable cells to be wholesale biosensors. But the notion of putting it in somebody's body was crazy. The idea of the microbiome was not prominent. Nobody was talking about it. And so we talked about how you could engineer these, place them on chips, computer chips, electronic chips, and place them in a quad at a university to see was there an anthrax release or was there detectors of lead paint in a home or on lead on imports. And showed that you could engineer these quite readily. And then by the late 2000s, early this decade, a lot of excitement arose. Could you engineer bacteria to serve as living diagnostics and living therapeutics? Could we engineer bacteria to detect and treat cholera? And we decided to engineer Lactococcus lactis, which is found in dairy products, fermented milks, and showed you could engineer it to eavesdrop on cholera by taking the quorum sensing system out of cholera and repurposing into L-lactase. And that you could then harness the natural lactic acid producing capability of L-lactase to treat or inhibit cholera infections. It's incredible that you were tackling from the very beginning, both the diagnostic and the therapeutic aspects. Did that not seem like biting off a lot? No, no, I mean, engineers, we probably take on too much. Engineers are famous for being able to focus, but do it with lack of focus in the sense that they can take on big problems and thus lack of focus, but take on those big problems and try to accomplish it. We thought for sure it would be fine, even though it's really viewed as two distinct fields. And we launched what became SynLogic. And in a matter of a small number of years, they now have multiple clinical trials. They're a public company, and they're using E. coli to treat different rare genetic metabolic disorders, to treat inflammatory bowel disease, and now to treat cancer. What are the engineering principles that you tend to think are the most useful in this crossover space that are kind of your north stars? I mean, part of it is that, like, you know, when one is trained as an engineer, you learn certain principles. Do you think bioengineers can take some of those existing engineering principles and bring them over? Or are bioengineers inventing new engineering principles? You know, it's an interesting question. I think that in many cases, it's more techniques and approaches than it would be principles. I tend to think of principles really being technically grounded notions that have established a relationship between certain variables or parameters that you come in. And so what's interesting is that a major challenge in engineering biology, and more broadly, specifically synthetic biology, is that we don't really know the design principles. That's right. And that we'll put things together, and they'll be motivated by electrical engineering, but recognizing that obviously we're not making it in electrical or electronic circuit. But in many cases, the circuits don't work at the level as well as we like or as we predicted. And I think it reveals that we don't have those design principles in that establishing the relationships, for example, that really guide electrical engineers. We're still, I wouldn't say haptazardly putting them together, but it's still very much trial and error. And it leaves great opportunities for getting after design principles. At present, I think for really the first two decades of synthetic biology, it's really been more engineering approach. And that is this idea that you can design a schematic for your circuit, find the right parts, put them together, and begin now characterizing both the parts and their functioning in a circuit, and iteratively improve upon the behavior. The engineer will go after it in a highly parallel fashion. So as he was doing the toggle switch he would explore two dozen possibilities simultaneously and do it relatively quickly and identify the one or the couple that gave him the closest to what he was going after And then he would expand around that after a couple weeks and do this iteratively and get through what could be basically many dozens and hundreds of possibilities Was that because of just a different approach or was the technology actually enabling him to do that for the first time? It was really, I think, more the approach. And the distinction is that the young biologists in my lab will instead pick one design, one embodiment of that design, and they're going to drill down in great detail over, instead of two weeks, over six weeks, realize that it didn't work, and then they're going to go and create the next design on the basis of that, drill down one by one. More serial. Serial, and they will have a much greater in-depth understanding of that one case. But it's going to take them 20 times longer to get to a functioning prototype. And so there is where there still is really a cultural divide on the approaches. Would they argue there's something lost when you do it simultaneously, the engineering-led approach? I haven't heard them say that it's lost. Because otherwise, like, why not? Yeah, you know, there would be interesting is that I do think they feel that they'd like to get an in-depth understanding. And again, it's now coming up against the culture clashes. The scientist is curious, wants to understand, is looking for discovery. The engineer wants to get it to work and doesn't necessarily care do they understand how it works, but that it works. You know, the scientist really wants to understand, and making it work is proof of understanding. But understanding is the real ends, and making it work is the means. Whereas for an engineer, making it work is the ends. Yes. And understanding is the means. Yes. And because both care about the ends, the means, actually, they care less about. Yeah. And so they're optimizing for different things, even though from the surface, they may look like they're doing the same thing. And I'll draw an interesting further distinction on level of comfort is that I've been in the sin bio world now for about two decades, and I have yet to get significant buy-in from the engineers in my lab for doing directed evolution. So you would think after you get your prototype that's working okay, but not at the level that you like, that you would be open to using one of the great techniques. Noble prize winning techniques. by Frances Arnold, who's considered part of our community, and she's marvelous and a great pioneer. They would use directed evolution techniques to optimize taking advantage of this beautiful feature of biological systems. It is completely foreign and anathema to the engineers in that they view it as cheating, that they want to be able to themselves retweak this system by design to get it to function the way you like. The chemists and the biologists in my lab much more readily are embracing directed evolution. That's kind of funny. And I think there's now going to be three prongs because, you know, the success of directed evolution over most rational methods, let's say protein design, enzyme design, something like that, it's just really hard to do that rationally. There's a third arm. You have rational design where maybe trying to use physics and quantum mechanics. The directed evolution where you just sort of optimize for some function. Machine learning is an interesting way to sort of have it both ways. In that, you know, from all the data you get from directed evolution, could you use that to actually come up with a predictor or readout? I do think machine learning within synthetic biology is one of the next frontiers. And the challenge there is a couple levels. One is can we have enough data to feed the models? And by there, the data is can we generate enough diversity and can we functionally characterize that diversity to feed the algorithm? Absolutely. I think the marriage of direct evolution with synthetic biology really is also the future, coupled to machine learning. Synthetic biology allows you to cheat in that it can move you in that genotype, phenotype space into an area that you can't get to readily through evolution. Can you break that down? If I'm going to tweak a system, whether it's naturally evolving or I'm going to use directed evolution to evolve it, you're really going to move incrementally. Now, at fairness, maybe you get a five-fold or even a hundred-fold or two-hundred-fold after many, many rounds, as Francis showed for enzymatic activity. But in cases still that you're going to be kind of starting in some place, say if you've got on your x-axis genotype and your y-axis phenotype, you're stuck over in this corner. Direct evolution will allow you to move from that corner, but you're going to stay in the general space. You're going to kind of randomly move around. Synthetic biology allows you to take your starting point far away from that corner. Or to hop. Or to hop over into another spot and then fluctuate around using direct evolution. And so it's the marriage of the two that allows you to explore spaces that would take hundreds of years, if not millions or billions, or what really has happened on the planet. Well, the interesting thing is you've seen— Sounds like wormholes to another side. Yeah, yeah, yeah. You've seen evolutionary approaches applied to electronic circuits, right, where they make a radio and it's just like some bizarre circuit that works, but it's hard for human beings to understand. It's funny, with biologists and engineers, our two different cultures, the machine learning is like a third one still, possibly. Do you think that science as a whole will be moving in this direction going forward more with this blend of engineering approaches? There's been a blending between science and engineering, especially in any of these interdisciplinary activities. I have Stanford colleagues that were trained as engineers but are writing science papers and doing science. And, you know, I'm trained as a physicist, but, you know, I've moved into engineering. And I think when you're at the interdisciplinary sort of interface, a lot of people do whatever it takes. Yeah. And so that spirit is there. Now, there will be people who stay in their domains that just love biology and are just doing biology, and that's all they want to do. And that's also fine. But I think the rise of the interface has really sort of led to this new area. I do see more and more of the interface between quantitative approaches in science, not per se engineering, but quantitative advances of really getting after data analytics and embracing this notion that machine learning and more broadly computer-based modeling and data analysis is becoming dominant as people are trying to figure out how to embrace our increased ability to collect a lot of data and analyze data. Yes, one thing that I was talking about internally and almost as an investing theme is, you know, where is synthetic biology on the hype cycle? You know, my read-on is it's gone past peak hype and is sort of in the now getting into production, getting too useful. Synthetic biology has been hampered by basically too much hype from its very beginning. And I think there's multiple reasons for that. One is that it's a very romantic, exciting field. This idea that you can engineer life to address some of the world's biggest problems is exciting. And second is that I think we've had an unchecked field that is not yet an established discipline. And so you have fringe elements that will grab their 15 minutes of fame to put out stuff that is frankly not realistic. Sounds right, likely will work, but could take centuries to accomplish. And we've been hurt by that. Now, if you go back to the mid-2000s, our field took a strong left turn toward bioenergy. And there are many factors driving it, starting around 2004, where you had gas prices going north of $4 for the first time. You had then President Bush calling for alternatives to petroleum. You had large oil companies looking for alternatives. You had venture capitalists saying, boy, there was a real opportunity here. And they heard about this exciting new space, Insthetic Biology, where people were engineering bacteria and fungi to convert different foodstuffs and sunlight into fuel. Sounds like a panacea. Sounds brilliant. And they poured hundreds of millions of dollars into it. You had Department of Energy and other national companies similarly pouring huge monies in, and it was too early. Too early in that you had brilliant academics showing that you could do this in small bioreactors in their lab at a university. But to think you could scale this up to the million liter bioreactor was absurd. And that you had loss of efficiencies. You just couldn't compete economically. And so many of these early companies got to be pretty good at producing $1 worth of gasoline, but it cost them $8 to do it. which was a horrible business model. Unless gas went up 10x. Unless gas went up 10x, yeah. And so that part of the field collapsed on itself on the hype. What happened then in the late 2000s when the bioenergy focus hit Earth, came back down, is that the field got grounded and really began building out more broadly tools, constructs, platforms, and turned toward biomedicine. I think the field got really regrounded and saw that there was great potential. And alongside that, you had other developments in biotech that were complementary to synthetic biology, broadly engineered cell therapy outside of SynBio, but like CAR-T therapy. And you then had the biotech discovery or development, I should say, of the century, which is on CRISPR, enabling very rapid, easy, inexpensive genome editing that together now opened up possibilities that just weren't going to be readily apparent to us 10 years ago. I mean, other directions that go for molecules that are just worth more than oil. I mean, a barrel of perfume is a lot more expensive than a barrel of oil. Yeah and so then the question is can you gain a lot by engineering a living cell to produce it versus using a chemist in a regular Petri dish Yeah yeah And that where I think the jury still out on many of these companies It seems like they can make it now the question is is there a business model there yeah i love to know about company building in this space what that looks like how is it actually different building a company in this space versus traditional bio and there's like two two models there's how to build a traditional bio company and how to build like an engineering driven tech company and this seems distinct from both you have to make a decision and that is are you a platform company or are you a product company? And what happens is that the companies in this space typically are being launched by the academic. As the academic comes in thinking they can do everything. And so they want it to be a platform company that's feeding a product company. And when you then talk to the real company builders, they'll say, no, we've got to make commitment. And I think they're hybrids. I mean, you can have these multi-product platform companies. Let's break it down what you specifically mean by that. So by that, I mean that on a platform company would be that I'm going to develop internal capabilities that will allow me, for example, to engineer bacteria to sense different things and produce molecules that would have an impact. To make many products. To make many, many products. And it might be that I'll be in focus on therapeutics, but that I could have the capability to engineer E. coli that would allow me to address 20 different diseases. And that my model then might be I'm going to team up and partner with pharma and large biotech. The product company would be, no, you know what I'm going to do? I'm going to engineer bacteria to go after your recycled disorders. and I'm going to launch a clinical trial and that's my product. I'm going to move it toward clinic. I could build and maybe have one or two other internal programs. But it's a struggle. In what way are you going to play it in this space? And you see companies that are playing both ways. Synlogic is really a product. It's a therapeutic, synthetic bodies therapeutic company that is focusing on developing therapeutic products that will go into patients and that they themselves, maybe through partnerships to market them, but develop them. Ginkgo, Bioworks, for which I'm not involved, is more of a platform company, developing capabilities to engineer organisms to optimize enzymes, and through a partnership model, could have a platform. The platform companies in this place are playing to the technology investors, and the product companies are playing more toward the traditional biotech investors. Although I think that's starting to change in that I think there's been cycles. So even the biotech investors are curious about platforms. I'm encouraged to hear that, right? It goes booms and busts. So when the NASDAQ collapsed 17 years ago in 2001, the local biotechs ran from platforms. Prior to that, from 98 to 2001, it was platform happy. And then they became fixated on our products to a fault in that many of the companies would rush a molecule into trials to say they had a product. And most of those molecules were horrible. And they ended up failing. The other thing that happens is that it's very tempting to, once you've built a platform and you actually have an asset that is, let's say, past phase one or phase two, that becomes worth so much. It's a hard financial decision to take money away from that and put it to other things. And then you become a platform company, becomes a single asset company. And I think that the model is that if you can move your assets, develop assets, move them into trials, you then also have that struggle. Do you now flesh out your platform? And I see that in companies I advise and I'm involved with. And I'm a big believer that you should also have the long view that it's worth building that internal capability, whether it's around design or whether it's around manufacturing. So, for example, having the ability to manufacture your product in-house significantly increases your value. What is the go-to-market like in this space when you're building these synthetic bio products or platforms? What is the way you see this kind of entering the system? How is that process happening? I think the challenge is really making a commitment as to what is your go-to-market. Because in many cases, these early companies, particularly coming out academically, are aspirational in that you want to take on all these problems. And yet, if you're really then going to get down, it's very hard to go to market and what's your choice. And then it really becomes specific. So in one area I'm involved with is diagnostics. Is it through a partnership model? Or are you really going to develop a product, get it to approval, and now introduce in the market? Well, that poses all fascinating new challenges of who's going to manufacture, how are you going to distribute, what are
