Optovolution: Teaching Proteins to Think Like Computers (EP. 31)

Hosted by Lester Nare and Krishna Choudhary, this episode is a deep dive into a new synthetic-biology breakthrough out of EPFL: OptoEvolution. The big idea is simple but powerful — traditional directed evolution is great at making proteins that are always “on,” but biology is full of proteins that need to switch states, respond to stimuli, and behave more like logic gates than static tools. This paper takes directed evolution and couples it to light and the cell cycle, creating a new way to evolve dynamic proteins that can toggle, compute, and respond with far more control. Summary Why directed evolution needed an upgrade — classic methods select for proteins with continuous function, not proteins that toggle between active and inactive states. OptoEvolution — using light as a control signal and the cell cycle as a built-in oscillator to evolve proteins that must turn on and off to survive. Color-multiplexed biology — engineering proteins to respond to different wavelengths of light, opening the door to finer control of gene expression. Single-protein logic gates — proof-of-concept AND-gate behavior inside a single protein, hinting at a future where biology can be programmed with much more software-like precision. Support the show Donate: FFPod.com/donate Follow: @FFPod on X / Instagram / TikTok / Facebook Show Notes OptoEvolution / dynamic protein control (Cell)

Speaker names marked ? are inferred from speaking time and the show’s known hosts — they are not voice-verified.

• 0:00Lester Nare?

  Hello, Internet. This is your captain speaking, Lester Nare, joined as always by my co-host and our resident PhD, Krishna Choudhary. Today, we are going to be touching on a protein evolution story in the lab directed by humans. So historically, if we've wanted to make proteins do things that we wanted them to do, we've used the process of evolution in order to do so. But there's a lot of limitations with that approach. This next story is taking that method and putting it on steroids, giving us the ability to now be able to turn these on and off

• 0:42Lester Nare?

  and sort of create almost like a computer, these simple logic gates that give us much more control. And it's fascinating how this is going to have implications in a variety of arenas. And the way in which they did so is very interesting. So we are going to learn about the science from the ground up today, because this is from first principles.

• 1:19Krishna Choudhary?

  So we want to make new proteins.

• 1:21Lester Nare?

  Yes.

• 1:21Krishna Choudhary?

  All the time, right? That's a lot of biomedical research going on right now. It's to make new enzymes, new types of proteins, to do all sorts of things. And traditionally, a lot of times, what we can do is use in-lab evolution to make novel proteins. Now, those techniques, for reasons that we're going to get into, only make certain types of proteins, specifically ones that always remain on. But if we want proteins with actual more natural dynamics, like proteins that turn on for certain things and off for certain things, or if we want to have like many logic gates, like the ones that we have in our computer for the and, or the or, or the exclusive or, things like that. It's been really difficult to use the traditional technique to build those types.

• 2:05Krishna Choudhary?

  This is taking that step forward, okay? It's a breakthrough called opto-evolution from EPFL in Switzerland, Lausanne.

• 2:15Krishna Choudhary?

  It's a cell journal publication and the mechanism is really, really cool. What it's doing is it's using continuous evolution with light to steer the evolution of dynamic proteins, not just proteins that are going to be in one state all the time, but proteins that can transition and be in dynamic states. It couples like effectively like an oscillator, like optical oscillator with internal cell cycle oscillators. There's like incredible like deep physics underlying this and it's, it's actually really, really cool. Um, and to get started, let's just go through some of the history of directed evolution. Okay. So evolution is really kind of an optimization, right?

• 3:00Krishna Choudhary?

  We all know it as nature's way of engineering biological systems. There's the idea of survival of the fittest, which is you've got some population inside of the population, inside of that DNA, there's a lot of variation when it goes from one generation to the next. So you have variations in the genetic code, which leads to variations in phenotype, which means that, you know, like I'm going to have black hair or blonde hair or whatever. And through natural selection, the organisms that function most effectively are going to reproduce more and they're going to proliferate more. And that's basically the central tenet for evolution. Now humans have harnessed this process for a very long time to do artificial selection, artificial evolution.

• 3:41Krishna Choudhary?

  Early farmers influenced evolution by choosing which crops or which livestock to actually move on to the next generation. And if you look at, you know, the thousands of years of selective breeding have given us all of the crops that we know and love, but they started out not great. This particular photo, the one on the upper left, that's watermelon. That does not look like watermelon. It does not look appetizing. That does not, no, right? Um, the one in the middle, that's corn on the right, that's a carrot. That looks like a stick. That looks like a twig. That looks like a twig. That's not what we see in carrots, right? Um, so. All of the food that we eat today is a result of artificially selecting the ones that we prefer and then having them proliferate to the next generation.

• 4:26Krishna Choudhary?

  So this is not a new thing for humans to sort of take over the evolutionary reins and steer organisms to their will. Okay, here what we're doing is at a biomolecular level, we're doing targeted directed evolution. Okay, this is the idea where you induce mutations in the DNA. So you make thousands of versions of an enzyme, let's say, from that DNA. And then you select the ones, the versions of that enzyme that do the best for the next round. So, you know, let's say I have a thousand different versions. I'm now gonna create thousands of different enzymes. Out of those, the top 100, I'm gonna select that DNA and then now do it again.

• 5:09Krishna Choudhary?

  Do a bunch of mutations, create a thousand different versions, again, test it. I do this natural selection at a genetic level now, right? At a molecular level. And what I'm doing is through that process, I can make catalysts, replace toxic chemicals. This has been huge for the biomolecular industry. We've created biofuels out of this. We've created agricultural chemicals. We've even created a better way to produce a drug for treating type 2 diabetes. So this has been around for a very long time. Actually, in the 1990s, there was a professor Francis H. Arnold at Caltech. She started doing this. She's really been a pioneer of this type of directed evolution.

• 5:53Krishna Choudhary?

  She won the Nobel Prize in 2018 for her work. Interesting tidbit about her, undergrad at Princeton. Woo! Yeah, it was an undergrad in mechanical and aerospace engineering, which I thought was interesting because from there she went and did a PhD at Berkeley in chemical engineering and then a faculty at Caltech. And I think this is just a really cool way to think about like... What you do in undergrad is not like gonna totally sequester you for the rest of your life. I think they say what past is not prologue or something? I mean, she did aerospace and mechanical and now she invented directed evolution and won the Nobel Prize in 2018 in chemistry, right?

• 6:34Krishna Choudhary?

  So it just shows that like in undergrad, really what you're trying to do is learning how to think, right? And that's what's important and then you can move on and use that skill later on to do whatever, whatever you want. It's not over yet. You still have a chance. Exactly. Um, so she pioneered directed evolution. Here's the problem.

• 6:55Krishna Choudhary?

  Directed evolution is all about static selection pressure. For example, if I want to make an enzyme that breaks down a certain toxin, what I'm going to do is create a bacteria with these, with these, with this DNA that is constantly inside this toxin bath. Okay? So now only those bacteria that have the enzyme to break down the toxin are going to live. But naturally, what that means is that the enzyme is going to be constantly on. It's going to be constantly trying to break down that toxin. It's never going to turn off. So it's gonna, this whole process of directed evolution is going to select for proteins that have continuous function, not dynamic function.

• 7:38Krishna Choudhary?

  That's very different from proteins that we see in life. Proteins are dynamic. Right. They move from one configuration to the other. Here, what we're seeing is a particular ligand, that green dot is moving in into the protein. And the alpha helix in blue is changing shape. The protein is literally changing shape and toggling between one state and another state. And this is very natural for proteins to do. They want to toggle between an active and inactive confirmation in response to some kind of stimuli. It's effectively acting like a molecular transistor in some sense, right? There's an on off switch. There's a zero and one. And we don't want a protein that's either always zero or always one. We don't want to be always asleep or always awake.

• 8:23Krishna Choudhary?

  Yeah, yeah, we want, we want things that actually wake up and do things based on their particular environment. And that's where this paper comes in. Okay? This paper. Is called light directed evolution of dynamic, multi-stable and computational protein functionalities. That's what they're doing. They're using light to do this directed evolution. And from that, they can actually figure out ways to create dynamic proteins. That's the opto-evolution solution that we have. And so, before we get into the mechanics of all that, let's do a little bit of housekeeping.

• 8:59Lester Nare?

  Yes. So, I think, I want people to understand how incredible it is to be able to have the opportunity to have this show and to talk to you about breaking science news stories every week. As some of you who watched our last episode may know, we are trying out a new format here, where we're going to have multiple episode drops per week, rather than one big monolithic episode. So you can expect us to have about two to three episodes out per week as we try out the format. Big story episodes like we're talking about today are focused on really getting deep in the weeds on all of the mechanics from first principles on a particular research paper. Occasionally, we'll do a deep dive in a general topic. We

• 9:43Lester Nare?

  have some of those coming up that are really exciting. And we'll always end the week with our SNL weekly weekend update style rundown, where we'll cover several stories, a little bit less in-depth, but more topics, because there's so much happening in the world of science every day right now. And we want to make sure we cover as much as possible. If that sounds incredible to you, that sounds like something that you want to see in the podcast universe. If you are not particularly motivated to listen to another talking head share the same opinion that the other talking head talked about, one of the best ways you can support the show is a like, a share, a comment, a follow. Bring it into the water

• 10:26Lester Nare?

  cooler at the office, at the lab, at school. Making sure that we are battling that billionaire algorithm is the best way for us to get this show out to as many people as possible. We are on our journey to build an ESPN for science in all ways. And all of the support that you are, audience and community, give us is the way in which we can continue to produce the show. Speaking of producing the show, it's just two of us, myself and Krishna. All this, the camera switching, the production value, the quality of the content is centered around two folks. And so one of the other ways you can support us is we do have a donation portal. If you would like to be a patron, ffpod.com backslash donate helps us monetarily support

• 11:11Lester Nare?

  this show and remain independent. And if, if you are interested in representing FFP Pod in the public, let us know that you'd like to see our merch store go live. We do have some interesting stuff ready to go. And we just want to make sure that there's interest in having FFP related clothing items, maybe a couple of special items that we will not talk about just yet. But with housekeeping over, we can jump back into the story, except for-

• 11:44Krishna Choudhary?

  And one more, because this should be coming out on Wednesday. So if anyone is at APS, at the APS Global Summit, we used to call it March Meeting, but now apparently it's called Global Summit. The American Physical Society Conference is the largest conference of physicists. I will be giving a talk on Thursday. If you're going, I think there's an app or, you know, you've got the program, look me up, Krishna Choudhary, I'll be giving a talk on Thursday. Come check it out. I am, you know, actively still doing stuff and trying to talk about it to other physicists.

• 12:17Lester Nare?

  It's very, very, very important and very, very, very important that you go there. Okay. I'm just, he's so embarrassed. I'll stop, I'll stop.

• 12:25Krishna Choudhary?

  No, I thought you were about to-

• 12:27Lester Nare?

  Go look at the previous episode if you want to.

• 12:31Lester Nare?

  And with that, again, we are so appreciative of those of you who tune into the show. And for, and a lot of you new folks who are coming in from our Netflix appearance on Dave Chang, welcome. We're gonna have a great time here. You might learn a little bit, you may not learn a little bit, but you still might find it interesting and entertaining. Yep. So let's get back to the story.

• 12:53Krishna Choudhary?

  We want to talk about opto-evolution, which is this new way that this paper has figured out how to use directed evolution, this idea of in-lab evolution, but now to create dynamic proteins, not just proteins that are remaining on, but can now toggle between an on/off switch, like normal proteins, okay? The physics that underlies the core principle is from coupled oscillators, okay? This is something that we learn about in undergrad physics. You've got two oscillators that are connected by some kind of mechanism. Over here, we see just two masses on a spring. And you can see, in this case, the energy from one is getting transferred to the energy of the other. And so one of them oscillates, and then the other one remains stationary.

• 13:37Krishna Choudhary?

  And then when the other one oscillates, the first one remains stationary. The idea is that if I were to apply this to my protein challenge, right? I want to create a protein that can oscillate between an on and an off state. So what if I can tie its performance to a fundamental oscillator of life, which is the cell cycle, the reproduction cycle of the cell, right? Because the cell has to grow, then it has to split, and then it has to grow, and then it has to split, and it has to grow, and it has to split. So if I could somehow couple the evolution of the protein in interest with that oscillator, such that it needs to oscillate

• 14:22Krishna Choudhary?

  in order to really talk to this fundamental oscillator of the cell cycle, then perhaps I can optimize for proteins that can be dynamic, rather than only proteins that can be on and off.

• 14:33Lester Nare?

  I want to ask a quick question, just to help me understand. When you say oscillate, can you opine on that a little bit in terms of what exactly you mean by oscillate?

• 14:45Krishna Choudhary?

  Yes, so in physics, when we talk about oscillate, we mean that it moves from two states in some type of way. It doesn't have to be continuous. It doesn't have to be discrete. For example, simplest oscillator would be a pendulum. It's going to the left, then it's going to the right, then it's going to the left, then it's going to the right. That's an oscillator.

• 15:08Krishna Choudhary?

  A mass on a spring, it's going up and then it's going down, it's going up and then it's going down. Um, a ball on a hill, a ball in a valley is going to the left, then it's going to the right, then it's going to the left, then it's going to the right. In physics, actually, it turns out everything is a simple harmonic oscillator.

• 15:22Lester Nare?

  Okay.

• 15:22Krishna Choudhary?

  And that's a harmonic in that like there's, I think, I think it just comes from like harmonies. And the fact that it's, you can fit it with a sine wave. And an oscillator, I think it just means it's oscillating from one state to another state.

• 15:35Lester Nare?

  Right.

• 15:35Krishna Choudhary?

  Right? And so the cell cycle is an innate biological oscillator. Because it's oscillating from growth to division, to growth, to division, right? So it's a cycle.

• 15:47Lester Nare?

  There's a cycle that has some kind of period.

• 15:50Krishna Choudhary?

  Yes.

• 15:50Lester Nare?

  Uh, and a, a starting position and an ending position.

• 15:54Krishna Choudhary?

  And then it goes back to the start.

• 15:55Lester Nare?

  And they repeat, there's a repetition of the movement between those two.

• 15:59Krishna Choudhary?

  Exactly. Yeah, yeah. And so, and so what we want to do here is in order to create an oscillatory protein, something that can toggle between an on and an off, I'm going to couple it, connect it somehow to the original oscillator of biology, which is the cell cycle.

• 16:17Lester Nare?

  And would the potential benefit of doing something like that be the biology? It's, it's sort of like the same reason we use, um, uh, was it quasars for clocks?

• 16:29Krishna Choudhary?

  Mm-hmm.

• 16:29Lester Nare?

  Uh, because it oscillates rhythm on a, on a cycle that is so consistent and so repetitive. It's a dependable system to base.

• 16:38Krishna Choudhary?

  Yeah.

• 16:38Lester Nare?

  Off of.

• 16:38Krishna Choudhary?

  Yeah, yeah. With a quasar, it's like so far away that it doesn't depend on my position, the, the earth's position around the sun. And so it's always in the same spot in the sky. Right? And so it doesn't depend if I'm in July or March, the quasar is always there. And so I can use that as my clock to figure out how long my day is. In, in this sense here, we're using the cell cycle because it's, the cell cycle is just going to go, right? Otherwise the cell dies.

• 17:04Lester Nare?

  Right.

• 17:04Krishna Choudhary?

  So. If I can make a protein that I need to be dynamic tied to the cell cycle, then if the protein arrests into either an only on or an only off state, it's going to kill the cell. And then that protein is not going to proliferate in evolution. That's the sort of high picture idea of how we're going to use evolution here in this case, right? Because now it's survival of the fittest, but in order to be fit, I need to be able to toggle the internal dynamics.

• 17:35Lester Nare?

  Understood.

• 17:35Krishna Choudhary?

  The protein needs to be able to go from one state to the other. Right? That's what's key. So let's talk about this biological oscillator that I've been going on about. This is the eukaryotic cell cycle, okay? Mitosis, which is something that I hope everybody who did high school biology knows about.

• 17:51Lester Nare?

  Yes.

• 17:51Krishna Choudhary?

  The idea of binary fission, right? The cell doubles its DNA and then it splits in two. And then it doubles its DNA and then it splits in two. Well, the doubling of that DNA happens during interphase, specifically the S phase. That's the synthesis phase. The splitting in two is the mitosis part. But after splitting in two, now each of these daughter cells have only one copy of DNA. In order to replicate again, they need to go through the S phase again. They need to synthesize the DNA and double their DNA and then split up again. Now, this entire cell cycle is driven by something called cyclin-dependent kinases, CDKs, okay?

• 18:30Krishna Choudhary?

  These cyclin-dependent kinases are formed and degraded at very specific times. And if we go back to photo 11, what we'll see is what's happening is

• 18:43Krishna Choudhary?

  a certain compound comes up during, let's say, the G1 phase, which is the very beginning of interphase. So this is right after mitosis, right after the split, a certain cyclin E shows up in the cell, okay? And that's going to trigger the next phase, okay? Then during the S phase, during synthesis, another cyclin comes up. That's going to trigger the next phase. Now, how is that triggering happening? The triggering is happening because the certain cyclin shows up and it immediately gets degraded. So it's only there for a short amount of time. So it's like this boop signal, you know? It doesn't stick around.

• 19:18Lester Nare?

  It's, it's maybe like, um...

• 19:22Lester Nare?

  Is an analogy, it's like the ignition when you're starting your grill. And you click the button on grill to get the ignition going.

• 19:29Krishna Choudhary?

  Yeah.

• 19:30Lester Nare?

  And it's only there at the beginning.

• 19:31Krishna Choudhary?

  Yeah.

• 19:31Lester Nare?

  But then the fire's going and it's not there.

• 19:33Krishna Choudhary?

  Yeah, yeah, exactly. And we don't want that, that, that, that, you know, the, the, the whole time, right? You want it to just sort of start and it starts the phase and then it keeps going. And how does the cell regulate that? Well, the cyclins show up as molecules and then they get degraded. So they're only transient, right?

• 19:47Lester Nare?

  Okay.

• 19:48Krishna Choudhary?

  The key here, and it'll make sense later, but the key for them was to find a cyclin, a particular sort of factor here that is like starting the cell cycle. That is essential for one part of the cell. But if it sticks around for too long, then it's going to completely destroy the cell cycle.

• 20:10Lester Nare?

  Interesting, okay.

• 20:11Krishna Choudhary?

  With the, with the starter that you have on the grill, it's gonna be annoying. The sound is gonna be annoying, but the grill is still gonna work.

• 20:17Lester Nare?

  Yes.

• 20:17Krishna Choudhary?

  We need to find a type of starter molecule that if it sticks around is going to be deadly. For later. And what they found is a cyclin CLB5. What this thing does is promote DNA replication. So essential, right? This is the thing that starts DNA replication for the cell. But it naturally gets degraded. And if it's still present, it's gonna lead to cell death.

• 20:38Lester Nare?

  Okay.

• 20:38Krishna Choudhary?

  Because of later on, it's just gonna mess with whatever, whatever is going on. It's not important like how it does it, but we just know if it sticks around, it's bad.

• 20:45Lester Nare?

  Yes.

• 20:46Krishna Choudhary?

  CLB5, okay? So they created a strain of yeast. The, the model organism here is yeast. They, they created a strain of yeast called Daj Bog. This is named after the Slavic sun deity, the god of the sun. And he is in charge of the day and night cycle. Okay, we're gonna learn about why it's called day and night cycle. Because what we're trying to do is use light in this entire process. So that's why they called it Daj Bog, okay?

• 21:12Lester Nare?

  Okay.

• 21:13Krishna Choudhary?

  Here's the trap that they're setting.

• 21:16Krishna Choudhary?

  When they made Daj Bog, which is this particular yeast strain.

• 21:21Krishna Choudhary?

  It has endogenous cyclins, right? That that are there because it wants to replicate. They deleted them all.

• 21:30Lester Nare?

  Okay.

• 21:30Krishna Choudhary?

  Okay, so now this yeast strain is completely dependent on what the researchers give it.

• 21:37Lester Nare?

  I see.

• 21:38Krishna Choudhary?

  Okay, we've held it hostage.

• 21:39Lester Nare?

  Yes.

• 21:39Krishna Choudhary?

  And now the starter thing, it doesn't have its own starter.

• 21:42Lester Nare?

  Yes.

• 21:42Krishna Choudhary?

  We are going to provide the starter.

• 21:44Lester Nare?

  So instead of the grill having the button on it where you can click it and it has the igniter that starts the fire, we remove the battery from that on the grill so it can't do it. And we're bringing in one of those long lighters ourselves and pointing it, but we control when it's going in there and when the light happens.

• 22:01Krishna Choudhary?

  Exactly. And now that lighter, that critical cyclin, CLB5, that we have, we're going to place it under the control of the protein of interest, the one that we want to evolve.

• 22:13Lester Nare?

  Yes, yes.

• 22:13Krishna Choudhary?

  Okay? And that protein of interest is going to act as a transcription factor for that cyclin. Why is this important? I told you that that cyclin is extremely dangerous if it's on all the time. Previously, the problem with directed evolution is that we were only getting proteins that were on all the time.

• 22:31Lester Nare?

  Yes.

• 22:31Krishna Choudhary?

  Now, that's going to cause cell death. That is no longer the fittest, right? In the evolutionary perspective, if this protein is on all the time.

• 22:42Lester Nare?

  Right.

• 22:42Krishna Choudhary?

  The cell's gonna die.

• 22:44Lester Nare?

  We we've basically created, it's it's it's it's like in the movies where there's a scene, we've put out a collar around the protein.

• 22:53Krishna Choudhary?

  Yeah.

• 22:54Lester Nare?

  And if it continues doing the thing we don't want it to do, the collar self-implodes.

• 22:57Krishna Choudhary?

  Yeah.

• 22:59Lester Nare?

  But we basically now have a means by which to prevent a type of protein, we don't want to persist.

• 23:05Krishna Choudhary?

  Yes.

• 23:05Lester Nare?

  From persisting.

• 23:06Krishna Choudhary?

  Exactly.

• 23:07Lester Nare?

  Isn't that, I mean, it's such a such a neat thingy.

• 23:10Krishna Choudhary?

  Thing.

• 23:11Lester Nare?

  And it's inserting itself because there's a natural process that's already happening.

• 23:15Krishna Choudhary?

  Yeah.

• 23:15Lester Nare?

  And we're basically trying to make a portion of the candidate proteins in the entire population not procreate.

• 23:22Krishna Choudhary?

  Yeah. Anytime there's a mutation.

• 23:25Lester Nare?

  Yes.

• 23:26Krishna Choudhary?

  Where this thing turns on and stays on, the cell's gonna die. Anytime there's a mutation and this thing turns off and stays off, it's gonna die. The only way the cell is going to survive is if my protein of interest, which is the thing that I'm trying to evolve, turns on, creates the cyclin, and then turns off to stop creating the cyclin, right? And now. This is the magic of the light.

• 23:51Lester Nare?

  Okay.

• 23:52Krishna Choudhary?

  The survival condition is the following, that protein of interest is, has a little chromophore, so it's sensitive to light.

• 24:00Lester Nare?

  Okay.

• 24:00Krishna Choudhary?

  I'm gonna turn it on with light. It's gonna turn on. And then when I turn off the light, it's gonna turn off. Okay? And if it ever goes against that signal that I'm giving.

• 24:13Lester Nare?

  It's, it's done.

• 24:14Krishna Choudhary?

  It's done.

• 24:14Lester Nare?

  So, so we have this external, we're basically trying to have the ability to say, to say, to, to control the state through the light as an external source of, of, of influence.

• 24:29Krishna Choudhary?

  Yes.

• 24:31Lester Nare?

  The, the internals are such that we've constructed it that it will always, it should, it, when it responds to our light, to our external perturbation, it will survive.

• 24:43Krishna Choudhary?

  Yeah. If it remains on when we've turned it off.

• 24:47Lester Nare?

  Then, then it'll die. And so, so now we've basically created a mechanism by which we externally can control the state of the protein via light as the mechanism to do so. Because if it no longer responds to our external probing, it will no longer survive.

• 25:05Krishna Choudhary?

  Yeah.

• 25:06Lester Nare?

  Meaning through the natural evolutionary process, we will kill off the ones that are not what we want to be there.

• 25:13Krishna Choudhary?

  Exactly, yeah. So now we're only keeping proteins that are toggling on and off.

• 25:18Lester Nare?

  On and off.

• 25:18Krishna Choudhary?

  And, and as long as the light is in sync with the cell cycle, which means, you know, it's like over, like, so it's gonna be like on for an hour, then off for an hour, on for an hour, then off for an hour. Because that's about, it's about, takes like two hours for yeast to go through the cell cycle and reproduce. And so that's what we're doing. We can drive this oscillator now with light. It's like you're, you know, pushing on a swing. And as long as the swing keeps going, you're good. But if the swing stays up. That yeast cell is gonna die.

• 25:46Lester Nare?

  Can I ask a quick clarifying question? You know, we've talked a lot about proteins on the pod before and why they're so important.

• 25:56Krishna Choudhary?

  Yeah.

• 25:58Lester Nare?

  Can we just briefly touch on, for folks who may not be familiar with why we would even, why this is even relevant, like having control over the protein's ability to exist or not exist is a powerful tool.

• 26:13Krishna Choudhary?

  Yeah.

• 26:13Lester Nare?

  But to what end, maybe?

• 26:15Krishna Choudhary?

  Yeah, sure.

• 26:16Lester Nare?

  Yeah, does that make sense?

• 26:17Krishna Choudhary?

  Yeah, so proteins are the workhorses of life.

• 26:21Lester Nare?

  Okay.

• 26:22Krishna Choudhary?

  We, when we think about life, we think about like DNA, right? A lot of times we think about genetics. The reason why genetics is important is genetics is the blueprint for creating the machinery.

• 26:32Lester Nare?

  Okay.

• 26:32Krishna Choudhary?

  Okay? You can imagine genetics, the DNA is the IP. But the protein is the car and the vacuum cleaner and the lights and the camera and the computer.

• 26:44Lester Nare?

  The DNA is the, the KFC 13 herbs and spices secret recipe.

• 26:50Krishna Choudhary?

  Yeah, the protein is the actual.

• 26:52Lester Nare?

  It's the chicken.

• 26:53Krishna Choudhary?

  It's the chicken, right? And so what we want to do, when we're, when we're doing this directed evolution here.

• 26:59Lester Nare?

  Yeah.

• 26:59Krishna Choudhary?

  In the KFC analogy, we're changing the recipes to create the best chicken, right?

• 27:04Lester Nare?

  We want wings, we want tenders, we want, you know.

• 27:07Krishna Choudhary?

  Yes.

• 27:08Lester Nare?

  Da, da, da.

• 27:08Krishna Choudhary?

  And so far with the directed evolution, the way that we've done it is, you know, the toxin's always been there. So this particular machine, the protein, has just always remained on. It'll always eat the toxin, it'll never not eat the toxin, and it's just always going gangbusters. That's not how a lot of proteins in life work. A lot of proteins will toggle between being active and being inactive, and they only get active when there's a certain stimulus. For example, the lactase enzyme, the lactase enzyme which digests lactose only gets on or only really is expressed when there is lactose in the environment. Now there, the difference is the protein isn't actually there in the first place.

• 27:51Krishna Choudhary?

  Until lactose is there, lactose then triggers the transcription of that DNA to create the lactase enzyme that then digests lactose. But here, we want to create proteins that. Can toggle while being present, right? We don't, we don't want to initiate their production. And then there's a time delay because the DNA has to be read into RNA, which has to be read into this and then become a protein. And then the protein goes right. What if we want instantaneous reactions from a protein? Well, we want it to be off and then we want it to turn on. That's a good question.

• 28:25Lester Nare?

  Yeah, yeah, okay.

• 28:26Krishna Choudhary?

  Does that make sense?

• 28:26Lester Nare?

  Yeah, no, it does. And so the justification for trying to research this area is if we can control the state of the protein, there are a variety of ways in which with that capability, we can try to pursue any number of, let's say, therapeutic outcomes.

• 28:50Krishna Choudhary?

  I mean, like, for example, even with just the old directed evolution, right? The reason why the 2018 Nobel Prize was given to this is because there's been tons of research and tons of pharmaceutical uses from just classic directed evolution. And that directed evolution was only giving us proteins that always remain on. Now imagine if we can have a dynamic protein that comes out of this. And later on, we'll get into something that's very, very cool.

• 29:18Lester Nare?

  That's helpful. Thank you for, I just wanted to re-ground really quick. Please continue.

• 29:22Krishna Choudhary?

  No, that makes sense. So that's the, that's the idea. That's the whole idea is that we're trying to use light in synchrony with the cell cycle. The light is going to drive this protein. And as long as this protein can turn on and then off and then on and then off, we're good to go.

• 29:38Lester Nare?

  Based on the input of the light being present or not.

• 29:40Krishna Choudhary?

  Exactly. Yeah. So now let's get into some proof of concepts. Does this work? Okay. The first thing we're going to do is spectrally modify the light oxygen voltage domains of a certain protein called EL222. This is a protein that is ubiquitous in research. Okay. It's a transcription factor. So what that means is this protein, I shine light on it. That's going to attach to DNA, whatever part of DNA that you want. And then that part of DNA is going to be transcribed into mRNA and it's going to cause the gene to be expressed. So we can turn on and off genes using EL222.

• 30:25Krishna Choudhary?

  I shine blue light of 450 nanometers.

• 30:30Krishna Choudhary?

  And whatever gene of interest is going to turn on.

• 30:33Lester Nare?

  Interesting.

• 30:33Krishna Choudhary?

  That's an incredible technology already.

• 30:35Lester Nare?

  Right.

• 30:35Krishna Choudhary?

  Okay.

• 30:36Lester Nare?

  Right.

• 30:37Krishna Choudhary?

  But it's only one color. Okay. What if I wanted to turn on two genes? Right? Well, you could say, well, I just get an EL 222 on gene A and a EL 222 on gene B. But then, when I turn on blue light, both are going to get expressed. What if I want to turn on one? And then maybe sometime later, I want to turn on the on the other. And then I want to turn on both later. I'd need two colors of light. Right?

• 31:03Lester Nare?

  So so currently, we sort of have a very naive way to start the factory.

• 31:07Krishna Choudhary?

  Yeah.

• 31:07Lester Nare?

  We can basically say, start the whole factory.

• 31:09Krishna Choudhary?

  Yeah.

• 31:10Lester Nare?

  What we're trying to say is we want to start the chip department only.

• 31:13Krishna Choudhary?

  Yeah, and then maybe a time later.

• 31:14Lester Nare?

  We'll get the phone department going because the chips are ready.

• 31:18Krishna Choudhary?

  Exactly.

• 31:18Lester Nare?

  Whatever it might be.

• 31:19Krishna Choudhary?

  Exactly. So let's get a little bit into this um this protein here, EL 222. Okay? A lot of these proteins that are light sensitive use something called a chromophore. Okay? This particular one uses a flavon mononucleotide chromophore. That's the FMN. If you look at the protein here, the bulk is the protein. And then that little gray, sorry, no, the the bulk of the protein isn't gray. The black little compound there that looks like a nucleotide. Nucleotide is the building block of DNA. In this particular case, that little black molecule needs to be there. That black molecule is going to absorb the blue light photon, go into a higher energy state, like in quantum mechanics, just classic quantum mechanics. It's going to go into higher energy state.

• 32:03Krishna Choudhary?

  The protein is then going to sense that higher energy state and change its shape.

• 32:09Lester Nare?

  I see.

• 32:10Krishna Choudhary?

  Okay? And for the longest time, that blue photon, which is at 450 nanometers, blue, remember, is high energy.

• 32:17Lester Nare?

  Yes.

• 32:18Krishna Choudhary?

  Which means that that chromophore, which is the little triggering molecule, requires a high energy photon to bump up to that high quantum state. And then that causes the shift in the protein structure. Some random alpha helix goes from one state to another state, and then that activates the protein. The challenge is. That chromophore is pretty happy with blue light. It doesn't want to accept other nanometer wavelengths, right? So shifting the absorption to let's say a lower energy green light, because green light, there's a bunch of lasers we have with green light. It would be really nice if we could make a chromophore that shifts to green light.

• 32:56Lester Nare?

  Okay.

• 32:56Krishna Choudhary?

  Okay?

• 32:57Lester Nare?

  Okay.

• 32:58Krishna Choudhary?

  How do we do that?

• 32:59Lester Nare?

  Yeah.

• 33:00Krishna Choudhary?

  Right?

• 33:00Lester Nare?

  Because we talked about purple lasers recently.

• 33:02Krishna Choudhary?

  Yeah.

• 33:03Lester Nare?

  And.

• 33:03Krishna Choudhary?

  But that's hard, right? Green light is easier because it's lower energy, so it's it's fine.

• 33:09Lester Nare?

  Right.

• 33:09Krishna Choudhary?

  Right? So I'd like to be able to create an EL 222 that doesn't just respond to blue light, but there's another one, there's another version that responds to green light. That way, if I want to start the chip department, I shine the blue light. Then if I want to start the phone department, I shine the green light. And I have independent control of which gene I'm going to turn on.

• 33:29Lester Nare?

  We want to have more granular ability to turn the factory on to generate the proteins we want to, under the circumstances we want to. And then be able to also stop that process when we want to. And then have this control of saying if the factory starts producing the thing we don't want it to do, the factory will automatically shut down.

• 33:48Krishna Choudhary?

  Yeah, yeah. And so I want like, I want, I want multiplexing ability. So here's what, here's what they did. They selected under green, orange and red LEDs. And again, you do the same thing. You do directed evolution. You shine it on green, those that survive, make it through, those that don't, oops. What they found was key mutations, they actually were able to create this almost impossible

• 34:17Krishna Choudhary?

  light oxygen voltage transcription factor. The chromophore didn't change, okay? The little black molecule didn't change.

• 34:25Lester Nare?

  The original triggering one we talked about.

• 34:27Krishna Choudhary?

  Didn't change. It turns out there's some intermediate state that that thing has that does respond to green light. It goes into an intermediate quantum state, but the original protein was not sensitive to that change. Through two changes in amino acids, there was at the 83, at the 83 location, T went

• 34:47Krishna Choudhary?

  So the threonine went to alanine. One amino acid was switched for another. And at the 80 location, the glycine was moved to arginine. Both of those 83 and 80, as you can see, they're very close to that black molecule. So they're right next door. And just those two changes made the entire protein sensitive to that intermediate quantum level that green light triggers. And now on the left, you can see now the yeast is growing.

• 35:15Lester Nare?

  Yeah.

• 35:16Krishna Choudhary?

  Using those.

• 35:18Lester Nare?

  Yeah.

• 35:19Krishna Choudhary?

  Changes. Right?

• 35:21Lester Nare?

  And this, this is what's kind of interesting here is again, you, you, you come up with the plan. You try it and then you find out, oh, actually there's this thing that we didn't understand, which is this intermediate state. That exists. And if your fundamentals are good, then you can then continue the process because then you're like, okay, well now if we manipulate these intermediate stages, we should still be able to get the results we're looking for.

• 35:46Krishna Choudhary?

  Exactly. And now, now we have a whole new EL 222 star, I guess. That is allowing for orthogonal color multiplexing.

• 35:56Lester Nare?

  Right.

• 35:56Krishna Choudhary?

  Now I can have green light. Now I can have blue light. Blue light on, green light off or vice versa. Right?

• 36:02Lester Nare?

  Well, cause, cause the, the combination of the two, it isn't, is its own.

• 36:06Krishna Choudhary?

  Yeah. Now I can, now I can control a bunch of genes. Imagine if I could do this over and over again, right? If I keep doing the directed evolution such that the bandwidth of the response on that protein is really narrow. So green light of this particular frequency is only going to trigger that protein.

• 36:24Lester Nare?

  Right.

• 36:24Krishna Choudhary?

  Green light of this other, you know, teal versus aquamarine is going to trigger some other thing, right? Cause with lasers, we can get really, really narrow bandwidth. We can be like, it is at exactly 450. It's not 451. It's not 452. It is 450 nanometers, right? So it, it again, like for biomedical research, this is huge. It's allowing multiplexing of genomes.

• 36:48Lester Nare?

  Yeah.

• 36:49Krishna Choudhary?

  Right? Of gene turning on and off.

• 36:52Lester Nare?

  Yeah. And, and, and to maybe distill it down to kind of a basic concept level. We're creating a controller.

• 37:02Lester Nare?

  For researchers to better drive the process, the research processes they want to around protein synthesis?

• 37:10Krishna Choudhary?

  Yeah, exactly. Yeah, and gene expression.

• 37:12Lester Nare?

  And gene expression.

• 37:13Krishna Choudhary?

  That's the big one. Yeah.

• 37:13Lester Nare?

  Okay.

• 37:14Krishna Choudhary?

  Yeah, and protein synthesis is gene expression.

• 37:16Lester Nare?

  It's the thing that drives the gene expression.

• 37:19Krishna Choudhary?

  Yeah, exactly.

• 37:19Lester Nare?

  Okay.

• 37:20Krishna Choudhary?

  Okay, so breakthrough number two.

• 37:23Lester Nare?

  Okay.

• 37:23Krishna Choudhary?

  Has to do with trying to use it with red light. Now with red light and infrared light, you can't use the EL 222 because red light is way far. You know, red light is at what, like 800, 700 nanometers, whereas this thing was at 450. So it's like almost half the energy.

• 37:37Lester Nare?

  Right.

• 37:39Krishna Choudhary?

  So you don't have any hope in using the same protein. You're going to use something else called the 5B PIF3 system, okay? This is something that uses 660 nanometers to turn on and 740 nanometers to turn off. Okay? Why would you want to use red light? Well, red light is very good for deep tissue penetration.

• 37:58Lester Nare?

  Yes.

• 37:59Krishna Choudhary?

  It's the same reason why sunsets are red. Okay, the sunset is red and the sky is blue because blue light scatters more because of Rayleigh scattering. And so that's why from every direction in the sky, you're getting blue light because the sun is illuminating it with all the colors. But really, the shorter frequencies are the ones that are, sorry, the shorter wavelengths, the higher frequencies are the ones that are coming to us from all different angles. But when the sun is setting, it has to go through a lot of atmosphere. So for the same reason, all the shorter wavelengths, higher frequencies are getting scattered away from us and the red light is penetrating through. Same thing happens in tissues, in biological tissue. If I want to do this kind of, you know, light directed, turning on and turning off

• 38:44Krishna Choudhary?

  of genetic factors and things like that in my biomedical research, but deeper in the tissue, the blue light is only going to get through so much. If I use red light or infrared light, that's going to get deeper into the tissue. And so it's very much to my advantage.

• 38:59Lester Nare?

  Have you seen the, speaking of red light?

• 39:02Krishna Choudhary?

  Yeah.

• 39:02Lester Nare?

  And getting deeper into the tissue, the new masks that have been created in sort of the, I guess, beauty and skincare space? And they're blaring red light at your face.

• 39:15Krishna Choudhary?

  Yeah.

• 39:16Lester Nare?

  Because it gets deeper in the tissue.

• 39:18Krishna Choudhary?

  Yeah.

• 39:18Lester Nare?

  I'm just.

• 39:19Krishna Choudhary?

  Same thing.

• 39:19Lester Nare?

  Same thing.

• 39:20Krishna Choudhary?

  Same thing, exactly. Now, these, these.

• 39:24Krishna Choudhary?

  These proteins, just like the EL222 that we were discussing with the blue light, these all rely on a chromophore, which is some kind of molecule that is going to absorb the light. That's going to get triggered into a high-energy state, and then the protein around it is going to be sensitive to that high-energy state and then change its configuration and become active. The particular chromophore here is Fyco cyanobilin PCB. This thing is hella expensive, okay? Um, a thousand, it's fourteen hundred dollars per two milligrams. It's also highly unstable, and it's not natively produced in yeast and mammals, right? So in order to actually express it in yeast, I have to give it this chromophore,

• 40:07Krishna Choudhary?

  and I have to give it the red light. It's just, it's just really expensive, and it's gotta be like timely.

• 40:13Lester Nare?

  Right.

• 40:14Krishna Choudhary?

  Because it degrades really fast.

• 40:15Lester Nare?

  Right.

• 40:16Krishna Choudhary?

  So the campaign that we want to do with this opto-evolution is can we make this thing respond to red light without that chromophore?

• 40:22Lester Nare?

  Without the like additional secondary piece?

• 40:24Krishna Choudhary?

  Exactly.

• 40:25Lester Nare?

  That is required to actually make the process.

• 40:27Krishna Choudhary?

  Exactly, without, without PCB.

• 40:29Lester Nare?

  Can we get the lobster tail without the caviar?

• 40:31Krishna Choudhary?

  Yes, that's exactly right. And so that's exactly what they did. They subjected these dashbox cells to red and far red pulsing and no external PCB. There were mutants that had loss-of-function mutations that survived and created and were actually responsive to PCB. But they had deletions of this particular enzyme, this particular gene called YOR1. Now, why YOR1? Because that YOR1 is basically a trash collector enzyme.

• 41:02Lester Nare?

  Okay.

• 41:03Krishna Choudhary?

  That's weird.

• 41:03Lester Nare?

  Yeah, yeah, yeah.

• 41:03Krishna Choudhary?

  Why would a trash collector enzyme

• 41:05Lester Nare?

  Right.

• 41:06Krishna Choudhary?

  cause me to be now sensitive to the red light that I was no longer sensitive for? I don't have the chromophore that I need for the red light, but if the trash collector is not there, all of a sudden I can be responsive to red light. What, what does that mean? That means the trash collector was collecting some other molecule that I was using.

• 41:24Lester Nare?

  Yeah, yeah, yeah, yeah.

• 41:25Krishna Choudhary?

  That I'm now using for the chromophore to be sensitive to red light.

• 41:29Lester Nare?

  Right, right.

• 41:30Krishna Choudhary?

  And now that the trash collector is gone, there's some random other molecule. That I can now grab and use to be sensitive to red light.

• 41:38Lester Nare?

  Yes.

• 41:38Krishna Choudhary?

  Isn't that cool?

• 41:39Lester Nare?

  Yeah, that is very good. The, the, the trash collector was taking away the active ingredient that allowed the response.

• 41:45Krishna Choudhary?

  Yes.

• 41:45Lester Nare?

  Red light. When you get rid of trash, like the active ingredients, then available.

• 41:48Krishna Choudhary?

  Then available. And that's exactly what they found. They found this active ingredient, biliverdin, BV. And when you give the yeast cell artificial BV, it'll respond to the red light. If you take out the trash collector Y O R one gene. It'll be responsive.

• 42:04Lester Nare?

  It'll also be responsive.

• 42:05Krishna Choudhary?

  It'll also be responsive. So now, what does that mean? That means that I, the endogenous biliverdin can actually trigger my red light receptor cells.

• 42:15Lester Nare?

  Right.

• 42:15Krishna Choudhary?

  All I have to do is delete the trash collector.

• 42:18Lester Nare?

  Right, right.

• 42:18Krishna Choudhary?

  Enzyme.

• 42:19Lester Nare?

  And this, this.

• 42:20Krishna Choudhary?

  And now it's way cheaper.

• 42:20Lester Nare?

  It's way cheaper. This goes back to what this thing you brought up before and continue to bring up, which is if we can use the body's natural system to drive the outcome we're looking for.

• 42:31Krishna Choudhary?

  Yeah.

• 42:32Lester Nare?

  Versus artificially inserting something, it is always better.

• 42:36Krishna Choudhary?

  It's always better. I didn't want to say always, but just in case.

• 42:39Lester Nare?

  Yeah. I just, you know what? You're, you're very correct. In biology, there's never an always.

• 42:43Krishna Choudhary?

  Generally speaking.

• 42:45Lester Nare?

  Generally speaking.

• 42:45Krishna Choudhary?

  You're right.

• 42:46Lester Nare?

  Yeah.

• 42:47Krishna Choudhary?

  It's better. Most of the time.

• 42:49Lester Nare?

  Most of the time, yes, yes.

• 42:50Krishna Choudhary?

  We'll leave.

• 42:50Lester Nare?

  Biology is a notorious.

• 42:55Krishna Choudhary?

  Well, actually.

• 42:56Lester Nare?

  Yeah. Biology is not like physics that way.

• 42:58Krishna Choudhary?

  But that's a really interesting point. Again, this and the tracing of the process to the root, to the first principle. And the why then allows you to make more calculated choices about how do you want to intervene or, or interact with the system to, to, to decrease the external.

• 43:16Lester Nare?

  Exactly.

• 43:17Krishna Choudhary?

  As much, as much external disruption as possible.

• 43:19Lester Nare?

  Exactly.

• 43:20Krishna Choudhary?

  Exactly.

• 43:20Lester Nare?

  This is, that's, that's, and the journey to get there, very clever.

• 43:24Krishna Choudhary?

  Yeah, yeah. And finally, breakthrough three, I think this is going to be your favorite.

• 43:27Lester Nare?

  Oh, my God, no.

• 43:28Krishna Choudhary?

  Okay.

• 43:28Lester Nare?

  There's another one.

• 43:29Krishna Choudhary?

  Yes.

• 43:29Lester Nare?

  Okay, so just a really good, so with the breakthrough number one, so.

• 43:32Krishna Choudhary?

  Breakthrough number one was we made the blue light sensitive protein.

• 43:36Lester Nare?

  Yes.

• 43:37Krishna Choudhary?

  Sensitive to.

• 43:38Lester Nare?

  Multiple.

• 43:39Krishna Choudhary?

  Green light.

• 43:40Lester Nare?

  Green light.

• 43:40Krishna Choudhary?

  Which was good because now and also in later, we can, we can be sensitive to, to multiple.

• 43:46Lester Nare?

  Right.

• 43:47Krishna Choudhary?

  Here.

• 43:47Lester Nare?

  Then now we figured out the, the, the red light trash collector piece.

• 43:51Krishna Choudhary?

  Mm hmm.

• 43:51Lester Nare?

  Uh, with the, the Raleigh scattering.

• 43:53Krishna Choudhary?

  Yes.

• 43:54Lester Nare?

  And now there's a.

• 43:55Krishna Choudhary?

  Now finally, the last one is single protein computers.

• 43:58Lester Nare?

  Okay.

• 43:58Krishna Choudhary?

  Okay.

• 43:59Lester Nare?

  Right.

• 43:59Krishna Choudhary?

  We are now trying to create an and gate.

• 44:02Lester Nare?

  Mm hmm.

• 44:03Krishna Choudhary?

  Okay, all the students of logic know about the and statement, right?

• 44:08Lester Nare?

  Mm hmm.

• 44:08Krishna Choudhary?

  I need both of my inputs to be. No, no, I need either my input zero or one to be one and I'll get, um. Am I showing? No, I'm showing a. This is, this is an or gate. Okay, guys, whoever's, whoever's looking at the, uh, um, thing, this is like a not and gate, right? Because the A and B, if they're one, this, the output is zero.

• 44:33Lester Nare?

  Yeah.

• 44:34Krishna Choudhary?

  So this is a NAND gate.

• 44:35Lester Nare?

  Yes, yes.

• 44:36Krishna Choudhary?

  Sorry, that, that's my fault. Okay. But in any case, an AND gate is when both the inputs are one and that's when the output is one. Otherwise, it's always zero.

• 44:44Lester Nare?

  Right, right, right.

• 44:45Krishna Choudhary?

  Okay.

• 44:45Lester Nare?

  Which the idea is like, yeah.

• 44:47Krishna Choudhary?

  This and that are true.

• 44:49Lester Nare?

  That means go.

• 44:50Krishna Choudhary?

  The total thing is true.

• 44:51Lester Nare?

  And prosper.

• 44:51Krishna Choudhary?

  Yes. Okay, so historically biological logic circuits, we can construct them, but usually what they require is a multi-gene cascading process. Meaning gene A is on and gene B is on, which means that gene C will be on. Okay, so it requires this and that to be on and both of them go and become that. I'd like all of this to be in a single protein.

• 45:13Lester Nare?

  Right.

• 45:13Krishna Choudhary?

  Okay, because that way it's not a slow, I don't have to wait for one gene to turn on another gene and another gene.

• 45:19Lester Nare?

  It's not a sequential process, it's becomes just an instantaneous.

• 45:22Krishna Choudhary?

  It's this protein.

• 45:23Lester Nare?

  Yeah.

• 45:23Krishna Choudhary?

  Right? And, um, it's not also resource intensive because to transcribe one gene, it's a lot of ATP to transcribe another gene, again, a lot of energy. And so what I want is a single protein advantage, the computation is instantaneous.

• 45:36Lester Nare?

  Yeah.

• 45:36Krishna Choudhary?

  Right?

• 45:37Lester Nare?

  Yeah.

• 45:37Krishna Choudhary?

  And there's negligible. Metabolic load.

• 45:40Lester Nare?

  Yeah, yeah, yeah, yeah.

• 45:41Krishna Choudhary?

  So what do I do? I use my tet on and tet off system. This is something that we have discussed in the, it was the time capsule.

• 45:49Lester Nare?

  Oh, one of my favorite. That one was so, yeah.

• 45:52Krishna Choudhary?

  That was a crazy episode.

• 45:53Lester Nare?

  That was a great. If you guys have not watched that episode, I think I put that in my favorites list for last year.

• 45:59Krishna Choudhary?

  Yeah.

• 45:59Lester Nare?

  Very fascinating.

• 46:01Krishna Choudhary?

  That was this year.

• 46:01Lester Nare?

  Wait, the time, oh, I'm thinking about, I'm confusing time, uh, time crystals.

• 46:06Krishna Choudhary?

  Yeah.

• 46:06Lester Nare?

  And, uh, and, uh,

• 46:07Krishna Choudhary?

  And this is the time capsule.

• 46:08Lester Nare?

  The honeycomb structure.

• 46:11Krishna Choudhary?

  Yeah, that one was also really good. But the idea is with, with, um, RTA, this is this tet on tet off system. Basically, whenever there's doxycycline, this, um, particular

• 46:22Lester Nare?

  That's right.

• 46:23Krishna Choudhary?

  Protein is going to get transcribed. The gene is going to get transcribed. And so what they did was they combined this tet on system, the RTA, with something called a pest degron, which is effectively, if you have this pest degron, it's targeting the protein for rapid destruction. Okay, so here's what I've done. I've created, um, a protein where if I get doxycycline, it's going to come on.

• 46:52Lester Nare?

  Yes.

• 46:53Krishna Choudhary?

  But as soon as it gets transcribed, part of it has this degron system to degrade. It's like a degrade flag. The proteosome is going to come in and degrade it. Okay, so it's only going to be transiently active for a very short amount of time. Okay, now, instead of being a continuous on and off, like a continuous thing that is either in an on or off state, now you're kind of gating it to a, a, a, a window of time that has an explicit start and explicit end. Exactly. And so here is why the logic works. Because it requires input A, which is transcription on from the light that I'm getting.

• 47:29Lester Nare?

  Yes.

• 47:30Krishna Choudhary?

  And input B.

• 47:31Lester Nare?

  Hmm.

• 47:32Krishna Choudhary?

  Doxycycline needs to be present.

• 47:33Lester Nare?

  Hmm.

• 47:34Krishna Choudhary?

  Okay, both need to be present and they need to outpace the degradation.

• 47:38Lester Nare?

  Hmm.

• 47:39Krishna Choudhary?

  Okay, if I turn off input, then the, whatever protein is still there is going to get degraded immediately.

• 47:46Lester Nare?

  Right.

• 47:47Krishna Choudhary?

  So now it's a super time sensitive.

• 47:49Lester Nare?

  Hmm.

• 47:50Krishna Choudhary?

  And switch.

• 47:51Lester Nare?

  Yes, yes, yes.

• 47:52Krishna Choudhary?

  Does that make sense?

• 47:52Lester Nare?

  Yeah, it does, it does.

• 47:53Krishna Choudhary?

  Right? It needs the and of the, of the light.

• 47:56Lester Nare?

  Yes.

• 47:56Krishna Choudhary?

  And the doxy.

• 47:57Lester Nare?

  Yes.

• 47:57Krishna Choudhary?

  If both of, and the light I can trigger immediately, right?

• 48:00Lester Nare?

  Right.

• 48:00Krishna Choudhary?

  The doxy can sort of just hang out.

• 48:02Lester Nare?

  Right.

• 48:02Krishna Choudhary?

  But the light I can now trigger immediately.

• 48:04Lester Nare?

  Right.

• 48:04Krishna Choudhary?

  And turn off, and as soon as I turn off, it gets degraded.

• 48:07Lester Nare?

  The, the light is our controller. The, the doxy cyclin is a part of the substrate. That's necessary to facilitate the environment where we have control. But we don't necessarily, as long as the doxy's present.

• 48:18Krishna Choudhary?

  Yes.

• 48:19Lester Nare?

  That's the only, that's the thing that matters.

• 48:21Krishna Choudhary?

  Exactly. And, and they were able to actually do this. They were able to actually create this and switch in a single protein. Now, just imagine, right? Like, computers are based on logic.

• 48:34Lester Nare?

  Right.

• 48:34Krishna Choudhary?

  On computational logic. And here, we're able to do computational logic with a single protein at, like, the second time scale resolution.

• 48:43Lester Nare?

  Oh, my God.

• 48:43Krishna Choudhary?

  Right? It just, it just opens up a lot of really cool avenues for biomedical research.

• 48:49Lester Nare?

  Yeah.

• 48:50Krishna Choudhary?

  For fundamental science research. Cause now, if we want to figure out what this particular gene does, we can use this and gate to be like, well, what is the logic of this particular gene in relation to the entire logic table of the rest of the genome?

• 49:04Lester Nare?

  It, it is, so would you say, there are a lot of ways you could utilize it. Is one way you could utilize it as basically, uh, being able to isolate in a very complex biological environment, certain things you want to look at and control it so that you can watch the process or the outcomes, knowing what the in, like, knowing that you're controlling, like, the inputs.

• 49:27Krishna Choudhary?

  The input, yeah.

• 49:28Lester Nare?

  Into the system?

• 49:29Krishna Choudhary?

  Exactly, yeah. Exactly. And from that, we can, as I said, like, create the logic table. And that really lets us understand how genes relate to one another.

• 49:36Lester Nare?

  Right.

• 49:37Krishna Choudhary?

  How they relate to external stimuli.

• 49:38Lester Nare?

  Right.

• 49:39Krishna Choudhary?

  Like, all sorts of stuff, right? And the idea is with this opto-evolution, we can now create proteins that have this dynamic behavior. That need a and, that are not always one. This is a logic. This is, this is one that requires inputs to turn on.

• 49:54Lester Nare?

  Mm-hmm. Which, which means then you could then, you know, have proteins that only turn on based on an external stimuli that we define.

• 50:07Lester Nare?

  And so, you know, if you're putting something in the body, you don't want it to be always be on. You want it to be on only when you want it to be on. And now we kind of have the early makings of a mechanism that we can repeat that is discreet, controllable.

• 50:22Krishna Choudhary?

  Yep.

• 50:23Lester Nare?

  And has also the ability to terminate at the end.

• 50:28Krishna Choudhary?

  Yeah.

• 50:28Lester Nare?

  Which is like, we talked about a story, for example, where it's you have the half-life of a drug in the body. And drugs can have a really long half-life, which goes through and but you can't really, you can't just decide, today, we want to turn it off.

• 50:42Krishna Choudhary?

  Yeah.

• 50:43Lester Nare?

  You kind of just need to let it, the system dissipate naturally. In this case, for this particular use case, we can say like, no, we're done.

• 50:50Krishna Choudhary?

  Yeah, yeah. And, and I just, I, the, the possibility of all the stuff that we can do with this is endless, right? So, so it's really, it's really like, like, when I say that, like, like, you know, right now, I don't have

• 51:04Krishna Choudhary?

  exact ideas about how this is going to be used, but that's kind of the beauty of it. It's so, it's such a new paradigm to take this oscillator, tie it to the cell cycle, and make it such that the protein toggles on and off.

• 51:16Lester Nare?

  Right.

• 51:17Krishna Choudhary?

  Right? Now, it's a whole new avenue of directed evolution that we can now use to create a myriad of different proteins, a space that we didn't even have access to before, right?

• 51:27Lester Nare?

  It would, it would have, would a, an analogy that makes sense be like, it's like when we first figured out how to use binary in computing systems.

• 51:38Krishna Choudhary?

  Yeah, like even before the transistor, right? The, the vacuum tube was big, was huge.

• 51:43Lester Nare?

  Right.

• 51:44Krishna Choudhary?

  Yeah.

• 51:44Lester Nare?

  Right. And we didn't know that we were going to get Uber or, or Facebook when we were starting to just be like, oh, we can like, have an on and off.

• 51:52Krishna Choudhary?

  Yeah, yeah. Yeah, imagine all the logic that we can now do, put in a cell, in a cell.

• 51:57Lester Nare?

  In a cell. We, we can have the same level of sort of software style engineering that we have in computers. Again, I know there's a lot of other details.

• 52:06Krishna Choudhary?

  Yeah, yeah, yeah. This will be more machine language.

• 52:08Lester Nare?

  Right.

• 52:08Krishna Choudhary?

  But.

• 52:10Lester Nare?

  As a, as a rough kind of reference point for people to try to understand who might not be, who might not grasp the concept as organically.

• 52:19Krishna Choudhary?

  Yeah.

• 52:20Lester Nare?

  We are building our own ability to create programs at a biological level.

• 52:25Krishna Choudhary?

  Yes, exactly.

• 52:25Lester Nare?

  Like the equivalent of software programs.

• 52:28Krishna Choudhary?

  It's the, it's, it's the beginning of that.

• 52:29Lester Nare?

  It's the very, very early, early, early, early stages.

• 52:33Krishna Choudhary?

  Yeah, it's exciting.

• 52:34Lester Nare?

  This is very, very good. And this was, this came out of, um, I want to make sure.

• 52:40Krishna Choudhary?

  EPFL.

• 52:41Lester Nare?

  That's right.

• 52:41Krishna Choudhary?

  That's the Ecol, something, something.

• 52:44Lester Nare?

  Ecole Polytechnique Fédérale de Lausanne.

• 52:46Krishna Choudhary?

  Yeah, there we go.

• 52:47Lester Nare?

  University de Bayruth, Bayruth, not Beirut, B A Y R E U T H.

• 52:53Krishna Choudhary?

  Yeah.

• 52:55Lester Nare?

  Not the, uh, anyway.

• 52:57Krishna Choudhary?

  Yeah.

• 52:59Lester Nare?

  Yeah, we gotta be careful of that.

• 53:00Krishna Choudhary?

  Yeah.

• 53:00Lester Nare?

  No, but, but this is, and again, you know, um, it's so, it's very, this was very dense.

• 53:06Krishna Choudhary?

  Yeah.

• 53:07Lester Nare?

  But I, I think it's again, the, the level of depth of previous knowledge and work. That created the base by which these researchers are now building on top of to try to move away from this previous directed evolutionary approach and trying to find a way to be able to create these control mechanisms within it. And the three different breakthroughs necessary.

• 53:38Krishna Choudhary?

  It was just, it was just proof of concept. They were just doing proof of concept. Imagine what other people are gonna come up with, right?

• 53:44Lester Nare?

  Right, right. And that's another interesting point is like, not all, people are sometimes like, well, not all, not all science is, has a consumer product at the end of it.

• 53:51Krishna Choudhary?

  It's just more science.

• 53:53Lester Nare?

  Right.

• 53:53Krishna Choudhary?

  But imagine the amount of more science we can do with this new tool.

• 53:57Lester Nare?

  Which will ultimately end up in real world.

• 53:59Krishna Choudhary?

  Yeah.

• 54:00Lester Nare?

  Impacts. This is very different than, uh, folks who, well, I was gonna, I was gonna talk about dark matter, which we'll never figure out and we'll never know in our lifetime. So that's one area where. To have real world impacts. Fascinating, fascinating story. We've had a lot of protein stories. I mean, we talked about the Yale Lego block story last year.

• 54:25Krishna Choudhary?

  Yeah, I mean, at the end of the day, when you get to the molecular level, everything is proteins.

• 54:29Lester Nare?

  Right, and we have to, we have to, we want to be able to do more with them. Again, we are in our new format on the show multiple times a week. You'll see us pop up in your feed on the podcast feeds. If you're on Spotify or Apple on the YouTube's and you'll catch some of our clips on the socials. This gives us more times to get deeper into each story. Instead of just 30 minutes and rushing through, we can really, really dive in and get deeper. We still have the rundown. The rundown will be a standalone. Catch that later this week and I am your host, Lester Nare, joined as always by my co-host and our resident protein synthesizer and PhD, Krishna Choudhary.

• 55:13Lester Nare?

  We will see you guys for the rundown.