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

0:00Hello Internet, This is your captain speaking, Lester Naray, joined as always by my Co host and our resident PhD, Krishna Chowdhury. 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, you know, 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 and sort of create, almost like a computer, these simple logic gates that give us much more control.

0:48And 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're going to learn about the science from the ground up today, because this is from first principles. So we want to make new proteins all the time, right? That's a lot of biomedical research going on right now is 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.

1:36Now, 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 mini logic gates like the 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. This is taking that step forward. OK.

2:08It's a breakthrough called Optoevolution from EPFL in Switzerland, Lausanne. 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.

2:43There's like incredible like deep physics underlying this. And it's, it's actually really, really cool. And to get started, let's just go through some of the history of directed evolution. OK, So evolution is really kind of an optimization, right? 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.

3:32And 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. 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.

4:01That does not look like watermelon. That does not look appetizing. That does not know, right? 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? 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. So this is not a new thing for humans to sort of take over the evolutionary reigns and steer organisms to their will. OK?

4:36Here what we're doing is at a biomolecular level, we're doing targeted directed evolution, OK. Now 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 1000 different versions, I'm now going to create thousands of different enzymes out of those, the top 100, I'm going to select that DNA and then now do it again, do a bunch of mutations, create 1000 different versions. Again, test it.

5:15I 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 is 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.

5:49She started doing this. She's really been a pioneer of this type of directed evolution. She won the Nobel Prize in 2018 for her work. Interesting tidbit about her undergrad at Princeton. Woo in Tigers. 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 what you do in undergrad is not going to totally sequester you for the rest of your life.

6:24I 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? 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.

6:48Exactly. So she pioneered directed evolution. Here's the problem. 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. OK, so now only those bacteria that have the enzyme to breakdown the toxin are going to live. But naturally what that means is that the enzyme is going to be constantly on.

7:24It's going to be constantly trying to breakdown that toxin. It's never going to turn off. So it's going to this whole process of directed evolution is going to select for proteins that have continuous function, not dynamic function. 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.

7:54The 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 0 or always one. We don't want to be always asleep or always awake. Yeah, Yeah.

8:23We want, we want things that actually wake up and do things based on their particular environment. And that's where this paper comes in. OK, this paper is called light directed evolution of dynamic multi state 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.

8:59So 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.

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11:44And one more because this should be coming out on Wednesday. So if you, 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 Chowdhury, I'll be giving a talk on Thursday.

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12:40Welcome. We're going to 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. We want to talk about optoevolution, 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. OK.

13:12The physics that underlies the core principle is from coupled oscillators. OK? This is something that we learn about in undergrad physics. You've got 2 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. 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 it's performance to a fundamental oscillator of life, which is the cell cycle, the reproduction cycle of the cell, right?

14:03Because 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 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. I want to ask a quick question just to help me understand. When you say oscillate, can you opine on that a little bit? Sure.

14:42In terms of what what exactly you mean by oscillate? Yes. So in, in, in physics, when we talk about oscillate, we mean that it moves from 2 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, 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. A ball on a hill, a ball in a valley, 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.

15:19In physics, actually, it turns out everything is a simple harmonic oscillator and that's it harmonic in that like there's, 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, right, 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.

15:47There's a cycle that has some kind of period, yes, and a starting position and an ending position. And then it goes back. To and they go, and they repeat. There's a repetition of the movement between those two. 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.

16:17And with the potential benefit of doing something like that be the biology, it's sort of like the same reason we use was it quasars for clocks, because it oscillates on a cycle that is so consistent. And so it's a dependable system to base off of. Yeah. With the quasar, it's like so far away that it doesn't depend on my position, the Earth's position around the sun and so on. 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.

16:54And 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, right? 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?

17:27Because now it's survival of the fittest. But in order to be fit, I need to be able to toggle the internal dynamics, understood? 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. OK? Mitosis, which is something that I hope everybody who did high school biology knows about. Yes, the idea of binary fission, right?

17:53The cell double s its DNA and then it splits in two, and then it double S 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.

18:29OK, These cyclin dependent kinases are formed and degraded at very specific times. And if we go back to to photo 11, what we'll see is what's happening is a certain compound comes up during, let's say, the G1 phase, which is the very beginning of interface. So this is right after mitosis, right after the split, a certain cyclin E shows up in the cell, OK, And that's going to trigger the next phase, OK. Then during the S phase, during synthesis, another cyclin comes up that's going to trigger the next phase.

19:04Now 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, it doesn't stick around. It's maybe like is an analogy. It's like the ignition when you're starting your grill and you click the button on the grill to get the ignition going. Yeah. And it's only there at the beginning. Yeah, but then the fire's going and it's not there. Yeah, yeah, exactly.

19:33And we don't want the, 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. OK, 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. Interesting, right?

20:11OK, with the with the starter that you have on the grill, it's going to be annoying. The sound is going to be annoying, but the grill is still going to work. Yes, 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 cycle in CLB 5. 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 going to lead to cell death because of later on. It's just going to 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.

20:46CLB 5 OK, so they created a strain of yeast. The the model Organism here is yeast. They they created a strain of yeast called Dash 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. OK, we're going to 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 Dash Bog. OK, here's the trap that they're setting. When they made Dashbog, which is this particular yeast strain, it has endogenous cyclins, right, that are there because it wants to replicate.

21:28They deleted them all. OK, OK, so now this yeast strain is completely dependent on what the researchers give it. I see. OK, we've held it hostage. Yes. And now the starter thing, it doesn't have its own starter. Yes, we are going to provide the starter. 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 yeah, yeah, yeah and pointing it, but we control when it's going in there. Exactly, exactly.

22:02And now that lighter, that critical cyclin CLB five that we have, we're going to place it under the control of the protein of interest, the one that we want to evolve. Yes, yes, OK. 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.

22:31Now 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, the cell's going to die. 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 a, a, a collar around the protein. Yeah. And if it continues doing the thing we don't want it to do, the collar self implodes. Yeah. But we, we basically now have a, a means by which to prevent a type of protein we don't want to persist. Yes, from persisting.

23:06Exactly. I mean, it's such a such a neat. Thingy thing, and it's inserting itself because there's a natural process that's already happening. Yeah, And we're basically trying to make a portion of the candidate proteins in the entire population not procreate. Yeah, Anytime there's a mutation, yes, where this thing turns on and stays on, the cell's going to die. Anytime there's a mutation and this thing turns off and stays off, it's going to 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 cycling, and then turns off to stop creating the cycling, right?

23:47And now this is the magic of the light, OK? The survival condition is the following. That protein of interest is has a little chromophore, so it's sensitive to light. OK, I'm going to turn it on with light. It's going to turn on. And then when I turn off the light, it's going to turn off, OK. And if it ever goes against that signal that I'm giving. It's it's done, it's done. 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.

24:29Yes, the the internals are such that we've constructed it that it will always it should. When it responds to our light, to our external perturbment, it will survive. Yeah, if it remains on when we've turned it off. Then it'll die. And 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. Meaning through the natural evolutionary process we will kill off the ones that are not what we want to be. Exactly.

25:14Yeah. So now we're only keeping proteins that are toggling on and off. On and off. 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 going to 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 me like 2 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 going to die. Can I ask a quick clarifying question? You know, we, we've talked a lot about proteins on the pod before and why they're so important.

25:57Yeah. Can we just briefly touch on for folks who may not be familiar with why we would even why this is even relevant, as like having control over the protein's ability to exist or not exist is a powerful tool. Yeah. But to what end? Maybe like or like. Yeah, sure. Yeah. That. Does that make sense? Yeah. So proteins are the workhorses of life. OK. 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. OK, OK.

26:34And 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. The DNA is the the KFC 13 Herbs and Spices secret recipe. Yeah, the protein is the actual. Is the chicken. Is the chicken right? And So what we want to do when we're when we're doing this directed evolution here? Yeah, in the KFC analogy, we're changing the recipes to create the best chicken. Right. We want wings, we want tenders.

27:06We want you know. Yes. And so far with the directed evolution, the way that we've done it is, you know, the toxins 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.

27:34For example, the lactate 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 1st place 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 want to initiate their production.

28:11And 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. Yeah. Yeah. OK. Does that make sense? Yeah. No, no, it does. And, and so the, the, the, 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 yeah outcomes. I mean, like, for example, even with just the old directed evolution, right?

28:53The 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. That's helpful. Thank you. I just wanted to reground really quick. Please continue.

29:22No, that makes sense. So 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 gonna 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. Based on the input of the light being present or not. Exactly. Yeah. So now let's get into some proof of concepts. Does this work? OK, the first thing we're gonna do is spectrally modify the light oxygen voltage domains of a certain protein called El 222. This is a protein that is ubiquitous in research, OK? It's a transcription factor.

30:04So 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 EL222I, shine blue light of 450 nanometers and whatever gene of interest is going to turn on. Interesting. That's an incredible technology already, Right? OK, right. But it's only one caller. OK, what if I wanted to turn on 2 genes, right? Well, you could say, well, I just get an EL222 on gene A and a EL222 on gene B, But then when I turn on blue light, both are going to get expressed.

30:54What 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? So currently we sort of have a very naive way to start the factory, yeah. We can basically say start the whole factory. Yeah. What we're trying to say is we want to start the chip department only. Yeah. And then maybe a time. Later we'll get the phone department going because the chips are ready, whatever it might be.

31:19Exactly. So let's get a little bit into this protein here, El 222, okay? A lot of these proteins that are light sensitive use something called a chromophore, okay? This particular 1 uses a flavin 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 bulk of the protein is in Gray. The black little compound there that looks like a nucleotide. Nucleotide is the building block of DNA.

31:48In 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 in a higher energy state. The protein is then going to sense that higher energy state and change its shape. I see. Okay. And for the longest time, that blue photon, which is at 450 nanometers blue, remember, is high energy. Yes, which means that that chromophore, which is the little triggering molecule, requires a high energy photon to bump up to that high quantum state.

32:28And 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. OK, OK, OK.

32:58How do we do that? Yeah. Right, cuz we talked about purple lasers recently. Yeah, but that's hard, right? Green light is easier cuz it's lower energy so it's fine right? Right. So I'd like to be able to create an EL222 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 chip department, I shine the blue light. Then if I want to chart start the phone department, I shine the green light and I have independent control of which gene I'm going to turn on.

33:29We we, 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. 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.

34:04Those that survived make it through. Those that don't. Oops. What they found was key mutations. They actually were able to create this almost impossible light oxygen voltage transcription factor. The chromophore didn't change OK, The little black molecule didn't change the. Original triggering one we talked. About 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 to A, so the thyro 9 went to alanine, 1 amino acid was switched for another, and at the 80 location, the glycine was moved to an arginine.

34:56Both 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 the yeast is growing. Yeah, yeah, yeah. Using those changes. Right. 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 stage that exists. And if your fundamentals are good, then you can then continue the process because then you're like, OK, well now if we manipulate these intermediate stages, we should still be able to get the results we're looking. At exactly and now, now we have a whole new EL222 star. I guess that is allowing for orthogonal color multiplexing. Right now I can have green light, now I can have blue light, blue light on, green light off, or vice versa. Right, because because the combination of the two it create it's it's own.

36:06Yeah, 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, right? Green light of this other, you know, teal versus aquamarine is going to trigger some other thing, right? Because 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.

36:42So again, like for biomedical research, this is huge. It's allowing multiplexing of genomes, yeah, right. Of gene turning on and. Off. Yeah, yeah, yeah, yeah, right. And, and, and to maybe distill it down to kind of a basic concept level, we're creating a controller for researchers to better drive the process, the research processes they want to around protein synthesis. Yeah. Exactly. Yeah. And gene expression and gene expression, that's the big one. Yeah, OK.

37:14Yeah. And protein synthesis is, gene is. What is what? The drive. It's the thing that drives the gene expression. Yeah, exactly. OK, OK, so breakthrough #2 OK has to do with trying to use it with red light. Now with red light and infrared light, you can't use the EL222 because red light is way far. You know, red light is at what, like 807 hundred nanometers, whereas this thing was at 4:50. So it's like almost half the energy, right? So you don't have any hope in using the same protein. You're going to use something else called the five BPIF 3 system, OK?

37:46This is something that uses 660 nanometers to turn on and 740 nanometers to turn off. OK, why would you want to use red light? Well, red light is very good for deep tissue penetration. Yes. It's the same reason why sunsets are red. OK, 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 the shorter wavelengths, the higher frequencies are the ones that are coming to us from all different angles.

38:23But 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 light directed, turning on and turning off 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.

38:56And so it's very much to my advantage. Have you seen the Speaking of red light and getting deeper into the tissue the the new masks that have been created in sort of the, I guess beauty and skin? Oh, I've seen those, yeah. And they're blaring red light at your face. Yeah. Because it gets deeper into. The tissue, yeah, I'm, I'm just same thing, same thing, same thing exactly. Now these, these 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.

39:44The particular chromophore here is FICO Cyanobilin PCB. This thing is hella expensive, OK, 1000, it's $1400 per 2 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 and I have to give it. The red light, it's just, it's just really expensive and it's got to be like timely, right? Because it degrades really fast, right? 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?

40:22Without the like additional secondary piece exactly that is required to actually make the process. Exactly without without PCB. Can we get the lobster tail without the caviar? 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 yor one.

40:57Now, why yor one? Because that yor 1 is basically a trash collector enzyme. That's weird. Why would a trash collector enzyme 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.

41:24Yeah, yeah, yeah, yeah. That I'm now using for the chromophore to be sensitive to red light, Right, Right. 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. Yes. Isn't that cool? Yeah, that is very good. The the the trash collector was taking away the active ingredient that allowed the response to the red light when you get rid of the trash like the active ingredients then available. 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.

41:59If you take out the trash collector YO R1 gene, it will it'll be responsive also be responsive. It'll also be responsive. So now what does that mean? That means that I've the endogenous biliverdin can actually trigger my red light receptor cells. All I have to do is delete the trash collector, right? Right. Enzyme and this this and now it's way cheaper. 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 versus artificially inserting something, it is.

42:35Always. Better. It's always better. I don't want to say always, but just in case, yeah. Just you know what? You're you're very correct. In biology, there's never an always generally speak. Generally speak right. Yeah, it's better. Most of the time. Most of the time, yeah. Yeah. Well, biology is a notorious well, actually, yeah, biology is not like physics that way. 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 is about how do you want to intervene or or interact with the system to to to decrease the external as much as much external disruption as possible. Exactly.

43:20Exactly. This is, that's, that's and the journey to get there. Very clever. Yeah, yeah. And finally, Breakthrough 3. I think this is going to be your. Favorite Oh my God no there's another one yes OK, so just a really good so the break breakthrough #1 so. Breakthrough #1 was we made the blue light sensitive protein sensitive to multiple. Green light, Green light. Which was good because now and also in later, we can we can be sensitive to to multiple right here.

43:47Then now we figured out the the the red light trash collector piece with the Raleigh scattering, yes. Now finally, the last one is single protein computers. OK, OK, right. We are now trying to create an AND gate. OK, all the students of logic know about the and statement, right? I need both of my inputs to be no, no, no. I need either my input zero or one to be 1 and I'll get am I showing? No, I'm showing a this is this is a OR gate. Okay guys, whoever is looking at the thing, this is like a not and gate, right?

44:29Because the A&B, if they're one, the output is 0. So this is a NAND gate. Yes, yes. Sorry, 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 1. Otherwise it's always 0. Right, right, right. OK. Which the idea is like, yeah, this. And that are true. That means the total thing is true and prosperous. Yes. OK 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, OK? So it requires this and that to be on and both of them go and become that.

45:10I'd like all of this to be in a single protein, right? OK. 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, it's not a sequential process. It's becomes just an instantaneous. Boom, it's this protein. Yeah, right. And it's not also resource intensive because to transcribe 1 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, yeah. Right.

45:37Yeah. And there's negligible metabolic load. Yeah, yeah, yeah, yeah. 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. Oh, one of my favorite that one was so yeah, that was. A crazy that. Was a great If you guys have not watched that episode, I think I put that in my favorites list for last year. Yeah, very. That that was this year. Wait, the time. Oh, I'm thinking about I'm confusing time.

46:05Time crystals, Yeah. And the. And and this is the time capsule, the the. Honeycomb structure, yeah. That one was also really good, but the idea is with with RTA, this is this TET on TET off system, basically whenever there's doxycycline this particular. That's right. Protein is going to get transcribed, the gene is going to get transcribed and So what they did was they combined this Teton 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.

46:43OK, so here's what I've done. I've created a protein where if I get doxycycline, it's going to come on, but as soon as it gets transcribed, part of it has this degron system to degrade. It's like a degrade flag. The proteasome is going to come in and degrade it. OK, so it's only going to be transiently active for a very short amount of time. OK, 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 window of time that has an explicit start and explicit.

47:20Exactly. And so here is why the logic works, because it requires input A which is transcription on from the light that I'm getting, yes, and input B Doxycycline needs to be present. OK, both need to be present and they need to outpace the degradation OK. If I turn off input then the whatever protein is still there is going to get degraded immediately right? So now it's a super time sensitive and switch. Yes, yes, yes, that makes sense. Yeah, it does.

47:53It does it. Does it needs the end of the of the light and the doxy? If both and the light I can trigger immediately, right? The doxy can sort of just hang out, right? But the light I can now trigger immediately and turn off, and as soon as I turn off it gets degraded. The light is our controller, the the doxycycline 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, yes, that's the only that's the thing that matters.

48:21Exactly. 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, on computational logic. And here we're able to do computational logic with a single protein at like the second time scale resolution, right? It just, it just opens up a lot of really cool avenues for biomedical research or fundamental science research. Because 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? It is.

49:05So would you say there are a lot of ways you could utilize it? Is one way you could utilize it as basically being able to isolate in a very complex biological environment certain things you want to look at and control it so they can watch the process or the outcomes knowing what the in like knowing that you're controlling like the inputs, the input, yeah into the. 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, right?

49:37How they relate to external stimuli, right? Like all sorts of stuff, right? And the idea is with this optoevolution, 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. Which which means then you could then, you know, have proteins that only turn on based on an external stimuli that we define. And so if you're putting something in the body, you don't want it to be always beyond. 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 discrete, controllable and has also the ability to terminate at the end, 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 go go through and but you can't really, you can't just decide today we want to turn it off. You kind of just need to let it the system dissipated naturally in this case.

50:48For this particular use case, we can say like, no, we're done. Yeah, yeah. And, and the possibility of all the stuff that we can do with this is endless, right? So it's really like when I say that right now, I don't have exact ideas about how this is gonna be used, but that's kind of the beauty of it. 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, right? 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?

51:27It would have would have an analogy that makes sense be like. It's like when we first figured out how to use binary in computing systems. Yeah, like even before the transistor, right, the the vacuum tube was big, was huge. Right. Yeah, 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 game. Yeah, yeah, that, that that's imagine all the logic that we can now do put in a cell in a cell.

51:57In a cell, 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. Yeah, yeah, yeah. This will be more machine language. Right. But as a as a rough kind of reference point for people to try to understand who might not be, who might not have grasped the concept as organically, we are building our own ability to create programs at a biological level. Yes, exactly like the equivalent of the.

52:27It's the, it's. It's the beginning of that it's. The very, very early, early, early, early stages. Yeah, it's exciting. This is very, very good. And this was This came out of I want to make. PFL. That's right. That's last. Time echo something something. Echo Polytechnic, Federal de la Sun. Oh, there we go. University de Beirut. Beirut. Not Beirut BAYREUTH. Yeah. Not the anyway, Yeah, Yeah, we got to be careful of that. Yeah. No, but but this is. And again, you know, it's so it's very. This was very dense.

53:06Yeah. 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. Yeah, necessary. It was just, it was just proof of concept. They were just doing proof of concept. Imagine what other people are going to come up with. Right, right.

53:45And 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. It's just more science, right? But imagine the amount of more science we can do with this. Neutral. Which will ultimately end up in real world. Yeah, impacts. This is very different than folks who, well, I was going to talk about dark matter, which we'll never figure out and we'll never know in a lifetime.

54:07So that's one area where the ability to have real world impacts. Fascinating, fascinating story. We've had a lot of protein stories. I mean, we talked about the Yale A Lego block story last year. Yeah. I mean, at the end of the day, when you get to the molecular level, everything is proteins. Right, right. And we have to, we have to be, 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 Youtubes, 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.

54:58The Rundown will be a stand alone catch that later this week. And I am your host, Lester Naray, joined as always by my Co host and our resident protein synthesizer and PhD Krishna Chowdhury. We will see you guys before the rundown.