I actually picked up my copy of the history of the biochemistry department to learn a little bit more about him. And this was a chapter written by Hector DeLuca, which he describes the discovery of the production of high-density and water and milk by radiating these materials with UV. And at the time, so this led to patents, and the university's stance on patents was that they were under the sole ownership of the inventory. That'd be really nice, but they're still true. But so apparently, Professor Steenbach deposited the royalties of this money in an account and kept it crewing until he found a mechanism that could be used that would be suitably used to reinvesting this money. And this led, as many of us know, to the discovery of work. And in which Professor Steenbach was instrumental. Well, what's interesting was that Professor Steenbach assigned all these patents to work. And then those patents generated quite a bit of income from the university, 15% of which was returned to Professor Steenbach. And after some period of time, he refused to accept any additional amounts of this money was used to create personal funds that led to the establishment of two really large additions onto the biochemistry building. It endowed several Steenbach professorships. And among other things, started a symposium fund, which we're here today to celebrate. And I can think of no one's work that better kind of holds true to what's Harry Steenbach did than Professor Arnold, who really blends fundamental science and applied science. I'll just read this. Professor Arnold is currently the dean of Barbara Dickinson, professor of chemical engineering and biochemistry at Caltech. She has a really interesting background that included some time at the Solar Energy Research Institute of Bolton, Colorado, which I think many of us are really interested in. Thinking about biohubriels, the only postdoc at Berkeley in Caltech, she joined the faculty at Caltech and has many honors of which I'll just name two. She's a member of the National Academy of Engineering and the Institute of Medicine of the National Academy. She served on some very interesting boards, including the Santa Fe Institute, which is a really neat place, and several corporations. At Caltech, her research is focused on understanding the principles of biological design, and in that context, she has used evolution to create and select new proteins, metabolic pathways, and cells themselves. And these materials have applications, biotechnology, medicine, and energy for biofuels. And today, she's gonna tell us about her work on involving P450, which is fantastically interesting science, and I'd just like to remind you, in case you run out a little early, after today's talk, we'll have a banquet in her honor in biochemistry in the atrium, to which everyone's invited. Professor Arnold. Thank you very much. I'm delighted to be here and to share some stories of laboratory evolution. It's my favorite design algorithm. It's employed me for the last 20 years and probably will keep me busy for the next 20, and it works at all scales. Everything in this room of any real complexity is a product of this wonderfully simple design algorithm that even a chemical engineer can apply. And I get to apply it to some pretty fascinating complicated biological systems, and I'll be telling you today and actually tomorrow about my favorite model system, which is a real, real insight on cytochrome P450. I'd have to warn you, I've been practicing biochemistry without a complete license. I did a couple of postdocs in biochemistry labs, but I've not been trained entirely in biochemistry. I've been an engineer by training, and so my language may come a little different to you, and I hope you'll bear with me on that. I'm gonna tell you about a synthetic organic chemist's dream. Actually, I did this talk a lot, I talked about the system a lot to organic chemists, because here's an enzyme that can do some chemistry that would make your tongue hang out if you were trying to activate CH bonds. This guy can take non-activated CH bonds, mostly hydrophobic small molecules, and insert an oxygen from a dioxygen into that bond, and do so at room temperature, atmospheric pressure, and ate the solution. And it's really such a great model, of course, and it shows up in all the textbooks of what biology can do, but no one would touch it with a tenth of a pole if they're actually going to do this reaction. So, yes, we can admire the ability of this complex system to do it, but we don't actually do it. Of course, the stoichiometry of the reaction requires some delivery of reducing ophthalates from NADPH, which, of course, costs about 1,000 times per mole, more than almost anything you wanna make with it, so you have to do a little bit of cofactor regeneration and other things to make such a reaction go forward, not to mention, in fact, most of these pre-450s are membrane-associated and pretty much give up the ghost any time you go and try to purify them and characterize them, so they're a real biochemists dream. You can spend a lot of time doing that. So I'm telling you about the laboratory evolution of these catalysts, with the goal of making catalysts that one could use in ordinary synthesis, but also with the goal of trying to understand where all these remarkable capabilities came from. If you go out into nature and look at the some straight ranges of these enzymes and where they are, one thing that's remarkable is that just about everybody's got one. Just about everybody except for E. coli, which of course makes it fun to work with E. coli. I don't have a lot of background reactions. And just about everybody's got one. You've got about 57 of them. Plants can have hundreds of cytochrome pre-450s. They're involved in the first line of defense in the erbiotic breakdown. They're involved in various biosynthetic pathways and chemical products. And if you go and look at the range of substrates that'll accept for this oxidation chemistry so that it's enormously diverse, tends to be very hydrophobic substrates, but you'll see everything from very complex structures down to very medium chain alkane. So somebody a long time ago figured out how to do these reactions. And we look at this bailing of enzymes that exist today. Go to this database, this analysis database has more than 7,000 known sequences that number grows exponentially. So last year I did this talk, it was 4,000 out of 7,000. And next year it'll be many more. One thing we can see right away is that somebody figured out how to do this chemistry along time and go. And through the process of mutation, natural selection, elaborated on this such that over millions of years, the 7,000 sequences that you could scrape off the bottom of your shoe or extract from the plants or wherever today have diverged all over place to catalyze these reactions. And it's quite remarkable if you think about it. They also diverged very much in their sequences so that you can have things that are 85% different and yet have essentially the same three-dimensional structure and catalyzed reactions on a very broad range of things. Of course, this is extremely frustrating. It's remarkable to think that all these different sequences can fold into the same structure, but it's very frustrating to those who would go to these natural solutions to the problem of CHR and activation and try to discern what's the sequence function relationship. So one thing we know about these sequences is that there's a very large neutral component to evolution. There's a lot of random change being made that has essentially no effect on a function or very small effects on a function. And embedded within this 85% change in the sequence of these 300 plus amino acid substitutions might be three or four that are contributing to the functional changes. And it's your problem as biochemists to pull those three or four or 24 out of the 344 and trying to understand why one sequence has its particular function that another does not. So these products of evolution are beautiful, inspiring, but extremely frustrating to reverse engineer. For that reason, I chose one particular family member to play around with at the laboratory and a lot of people asked me, how do you choose a starting point? Well, I took advice from scientists at Nova Zan some years ago, they said, Francis, the last thing we want is an enzyme that you can evolve all over the place, which you can't make in train load quantity. You have to be able to make enough of this stuff to do something with, to understand so that you can do the crystallography so you can sell it, you name it. And so the reason that I chose to work with cytochrome P450 from the Silas Mediterranean is that you can scrape this organ, some of these are soil organism, off the bottom of your shoe, and you can put this genetic E. coli and make God want you to study. It's also, so it's well expressed in E. coli. It's a soluble version. It's sitting in the cytochrome class. It's not in the right position, like almost all the eukaryotic, like all the eukaryotic cytochrome P450s. Yet it's a P450 that shows a considerable structural homology to eukaryotic P450s. Even better, it's highly active. On its preferred substrates, this has a rate of up to 10,000 times the rates that the eukaryotic enzymes exhibit on their substrates. So we're starting with something that's good, that you can name in the plots of up to 12% of the cell protein will be the P450. And furthermore, unlike most of the eukaryotic, in fact, all of the eukaryotic enzymes which are multi-component systems, this has all catalytic machinery it requires to carry out these reactions encoded on the single-coli peptide chain. So the redactase domain, which applies three cofactors, is used to the heath domain and everything's there together in the single-coli peptide chain. So the question, oh, so what does this do? Of course, there's gotta be some downside of things. What it does is it hydroxylates fatty acids in subterminal positions. The action of wild interest unless you're making new detergents, of course that can be of some interest. And it doesn't show particular selectivity in its hydroxylation. So I think, for example, insert oxygen in any of the subterminal positions all the way up to about N minus five. And it usually inserts only one of those conversions. So here's the question. We have existing today in this family of thousands of known sequences and of course there are many, many thousands more that haven't yet been discovered. And I've identified people at 50 PM3, which hydroxylates fatty acid, yet I know that all these others are possible in fact there are many more. Could we use laboratory evolution methods, if these are powerful design methods, to create the three versions that exhibit any of these known activities? Can we start with a specialized enzyme today and create the functionally equivalent of all the other people? This is an interesting question because I can do my experiments in such a way that I'm making only adaptive mutations. And therefore I can ask, well what is the minimal number of evolutionary steps that it takes to go from one function to the other? And what are the kinds of mutations that will confer function to this? Are they occurring in the active site in the substrate binding pocket? Are they occurring somewhere else in the protein? I can actually follow the entire fossil record of an evolutionary experiment in the laboratory and see what it takes to achieve these new properties. Very interesting. I can go on and on, at least where we've discovered in-natural systems and ask, are non-natural activities accessible to this protein? And I'll give you an example of this. It's quite interesting to be able to probe the limits of the catalysis separate from what biology cares about because if we're biocanus and studying the natural world we're limited only to the products that make the natural selection. But there's a whole world of other products out there that might be the physically possible things.  I'll come back to this in a minute. Now one approach to making such proteins from the entry would be to sit down and try to design that. And everybody says, a test of my knowledge is that if I can build it, I understand it, right? So they tell you, if I've learned enough about protein folding and sequence function relationships I should be able to take a structure. The gene domain structure is known and identify the amino acid substitution that will convert it into some other function. That's hard to do. And I'm not going to go into any arguments although it's a wonderful debate that I often have with my colleagues in Caltech. I'll just tell you that in an enzyme like this details really not, if I call it enzymes, details really not. Catalysis, if you look at the energetics of the improving of, how does this tend to fold? We're taking only a few kilo calories per mole equivalent of a couple of hydrogen bonds out of hundreds that might be forming. And you have to be able to do your predictions of that level of detail in order to be able to design the catalysts. We simply don't understand those details. And luckily for us, we have an algorithm that circumvents this profound ignorance and allows us to do designs in the same way. But before I go in and tell you how I can evolve novel functions, I'd like to also remind you that evolution is not easy either. There's plenty of ways to make mutations for protein that give you nothing at all. Nothing of interest. A protein is less functional than what you started with. And I think just to give you a feeling of what the challenges are, it's useful to think of all the ways that you can string together the amino acids that make up a cytochrome P450 Hing domain. It's 450 amino acids long and in 20 letters in your alphabet, they've got a space of possible sequences that's not even remotely physically possible. This is a space that's so large that then it calls it very much more than astronomically. In fact, it's very much more than economically, which is the biggest numbers that humans can conceive of. It's very much bigger than any of those numbers, bigger than the number of particles in the universe by many four different words, magnitude. So this is a space of possibilities that nature has explored by the tiniest fraction of. And I can tell you I would explore it myself and even tiniest fraction of that, an infinitesimal fraction. Yet I will categorically tell you that most of it's empty. Most of those sequences don't encode for anything that even folds into a three dimensional structure much less a new cytochrome P450. And therefore it's a pretty hard place to be doing a physical optimization. Just give you some of the numbers and arguments. Experimental studies have gone in estimating what's the density of functional proteins in this sequence space. It's going from something like one in 10 and 12. Jack shows that pulling binding proteins out of random amino acid polypeptides of 90 amino acids to dug-axes in carbon-fated lacrysis in the creation of cytophings one in 10 and 70. So it doesn't really matter, you know, my intent is 12 is pretty small and you're trying to find the NEOs and K-sector antennas. 77 is pretty small, but all smalls of these things obviously did not come about why selection for random libraries. But of course that leaves us with a question of where did natural proteins come from? Had it obviously came from smaller things being built up, but that's under this lecture as well. And it should make us wonder, well, you know, if functional proteins are so rare in this space, how on earth do they evolve? Because one fact of life is that most mutations indeed are deleterious. If you go and make random amino acid substitutions for proteins and you ask the second question, what fraction of those retainability fold in function? So that's our new function, we just stay folded because we think that that's a prerequisite for function. Then that fraction changes exponentially down with amino acid substitutions, such that even after a very small fraction of amino acid substitutions, five out of four out of 50, maybe one in a hundred or even fewer would still fold in function. And then such a smaller fraction of those will acquire new functions, so it looks pretty ugly. A lot of biochemists and chemists and engineers will think, oh gee, proteins are fragile, right? So I can't make amino acid substitutions for them. But obviously that's not true because we are the products of evolution. We are the products of the process that has gone through the evolution. And really what the number to get from this is not that it goes down exponentially, but that there's a really significant number that are neutral. If this is the fraction that are neutral and we have an exponential function with m being the number of amino acid changes, we're talking of a fraction on the order of a half. And when you think of all the ways that you can make a single amino acid substitution to a protein that's 450 amino acid long, that's 19, that's 45, you're almost 9,000 ways, then a half times 9,000 still gives you many thousands of ways to make a mutation to a protein and still have a folding function. So it's this very large neutral fraction that allows us to move forward even if we're requiring new capabilities. John Maynard Smith in 1970 published a single 1.5 page long paper in Nature that I would most like to have written myself. And basically he argued that I believe we are the products of evolution and evolution has a certain nature such that for evolution to occur, I can tell you something about the distribution of functional proteins in sequence space. And in fact, the distribution has to be such that each protein is surrounded by one mutant neighbor and there has to be at least one next to it for evolution to continue its walk through space. So if we think of sequence space as being all proteins, functional proteins, and it's one mutant neighbor, so now they're ordered in this space with one mutation separating them, then mutation in natural selection diffuses walks one or two through this space finding new functional proteins nearby. And after a long period of time, they have diffused across this network so that the 7,000 cypro-p450s in 2008 will be related to one another eventually through all these single-mutational walks. And this tells us something that functional proteins today are embedded within networks highly dense networks compared to the rest of sequence space of functional proteins. So it doesn't matter where there's one in 10 to the 77 of functional proteins in a random space. What matters is that the products of evolution are sitting in a very functionally dense part of this space because that means that I can walk on this network too. All right, so go back here. See what it creates like because that tells you what I actually do at the top. So that means that I can actually use this idea and implement it in the laboratory through a very simple algorithm. If functional proteins are surrounded by other functional proteins through unit-mutational walks, then I should be able to take N18 from 2008 incorporate a few, a small number of random mutations into it, maybe one or two amino acid substitutions per gene. I could make lots of those in a test tube. Put those back into cells. They do the hard thing of translating that DNA into protein. And then, in front of cells, I should be able to screen for the properties of interest or selective. I'm smart enough to be able to tie that to survival and reproduction. So even if I do a simple screen, then of course the Murphy's law of evolution, so that's the majority don't do what you want. But if you ask the right kind of question where unit-mutational walks get rise to incremental changes in function, then yes, there will be some small subset in the order of 10 or five. And I can take those and repeat the process in a ranking on this, and essentially doing a very simple, stupid optimization, a random uphill walk in this fitness plan of state. And at the end of the process, either the student wants to graduate or you fulfill the milestones for your funding or you actually solve the problem, then the gene will encode a protein that gives the properties that you're interested in. This is a remarkably robust strategy, and I'll give you a couple of examples of the kinds of problems that you can solve with it. All right, of course, as opposed to that diffusing across the network for millions of years in the psychopathy for 50s, my walks in the sequence space because our patients is limited are very brief. Maybe we can do five, 10 generations. I'll tell you experiments of 23 generations, but these are very brief and they're very directed as well. This is by any means evolution, as we normally think about it. This is selective breeding, selective breeding of molecules in an ivory tree.  So what possible interesting function would be accessible by such a simple algorithm? Well, here we can use chemical information. If it were limited to a single mutation in every generation, and I have to have a measurable increase in oxygen, then we would imagine that some sorts of things that might be accessible to the M3 starting with the fatty acid hydroxylase activity, perhaps I could turn it into a alkane hydroxylase. And those would be structurally close so that a single mutation would get a measurable improvement in function. And then if I actually wanted to do something really interesting, such as confer an activity not exuded by the parental enzyme, then maybe I'd have to divide that problem into a series of steps, each of which could be solved by unidivitational rocks. So perhaps Isaac Kettis could say, okay, if I, let's say I wanted to do something crazy, and maybe people would say it works on methane and third oxygen into methane, where I'm starting with the fatty acid, then I can go through a series of intermediate steps, each of which can be solved by unidivitational rocks. Of course, you might come along and say, well, that's really stupid because it's not just the size of the substrate that matters, now you're actually getting into chemical difficulties here because we're also dealing with significant changes in the CH1 strength as we go to something like methane. So we're going from these internal methylene CHs all the way to about 105 kPa per mole for methane. So we're dealing with substrates that maybe people with 50s couldn't handle. And in fact, if you go to nature, you would find that there is no known natural occurring cycle of people with 50 that would take or is known to take gaseous alkenes or propane ethane or methane. Some people might interpret that. You see, I haven't been able to bring my P450 work on propane and I know of no propane oxidase that has a P450. Therefore, it cannot be done. That's a common response to such things in the literature. But I think that's going to be an interesting question because just because we have to get found with P450, in fact, that probably seems to be solved by a whole other class of enzymes that are not coming from oxygenases. While it might be tempting to say it cannot be done, maybe it's that nature never really had a reason to do it. So this nature solution to oxidizing ethane propane or diurnal enzymes that have no evolution or more mechanistic relationship to the psychrome people of this. This problem is solved by a very different and very complex family which I won't go into. But it's another family you don't want to touch with it and go, oh, isn't it very difficult to work with? And very difficult to engineer. So this is where I get into this question about what's fun about doing evolution in the laboratory is that, yes, if you want, you can limit yourself to the molecules that exist today or perhaps existed in the past because those are biologically relevant. But there's a whole other space of molecules out here that are physically possible at any time. That's a lot of places. It might be the cancer or the energy crisis out there. And they're just not exhibited in nature because they don't solve a biological problem. But it would solve my methane oxidation problem if I were able to solve that. Nature chose a different solution apparently. But it would be nice to know whether if P415 propane hydroxylase or ethane or methane hydroxylase is a physically possible molecule. If it is, then that brings another question. Why is this nature apparent in Q of the MMO? Okay, my part suggests we haven't looked very hard with gaseous substrates. A lot of microbiologists don't really like working with gaseous alkanes and perhaps there's not a big effort in screening P450s for these functions, but they exist. All right, so how do you actually do this? You take both fashion analytical chemistry and scale it down to 96 well plates and Matt Peters of chemist in my group did a wonderful job with that and came up with high throughput screens that give you enough information on whether something will take a substrate the size of octane or propane. Of course the CH5 strength is very different in these but at least it gave us a handle on the substrates. Well by the way, the reason you can't strain on octane or propane is it's really hard to measure octanol and propanol in a complex for a mutation problem. You don't want to purify your proteins, you don't want to do a lot of expensive steps because that gives you a lot of noise in your screening. You just want to take the lysate, add a substrate and get a nice color change. So if you take one of these methyl ethers, say di-methyl ether, then formaldehyde is the product of the P450 reaction and formaldehyde in purple reagent will form a nice purple color and light up with that reaction goes forward. Then you can take the product, the positive clones that are glued on di-methyl ether, test them on hydrogen gas chromatography and propane. So instead of the 3,000, maybe 3,000 here and then you can do 10 on gas chromatography and that's what we did and it used increase in the total turn number number that is how much product you actually make with propanol and that would be your sole criterion for accepting mutation. So I will make a long story short, this project started back in the 1990s with funding from the British Petroleum and they lost patience one way or the other to probably end this when we keep going with it. After, so with the white-type enzyme, it has a little bit of activity on octane and that's always a first principle of doing improvement by evolution. You really want to have a little bit of an activity to start with so that you're not just praying that the result will happen and actually have some belief that the mutation will give an improvement. So after five rounds of, and there's no active in propane, after five rounds of this evolution process we obtained the first decent alkane hydroxylase and it had improved activity on octane and along with that came some promiscuous activity. Once it had learned how to have reactivity on octane it also accepted a little bit of propane and made propanol and of course that was nice because we could then screen on that either or propane activity and when you screen this propane activity you can get large improvements of activity on propane and along with those large improvements in propane activity also come further improvements in activity on octane. So now we're up to five or six thousand turnovers, total turnovers and this is a manifestation of the most important law of direct evolution which is you get what you screen for. Good thing stopped, it was really not fun and I thought of many, you know, the students who had the project really didn't have the skills to do the screening now, we were chairing our hair up, just cut them for improvements and this often happens with these experiments. Why would things stop? Well, why would it stop? Because for instance, Cyclone P450 really does not want to do any better hydroxylating propane, certainly not ethnic, that's possible. Maybe that property is not chemically accessible by, it's not chemically possible, but the other thing is that if it's not accessible by this, it might be chemically possible, but it's just not accessible. And what turned out to be the issue was a really simple thing. The reason it wasn't accessible by an indentation of law is that you get what you screen for. We have been screening just for improvements in total turnover now. Does it oxidize more propane to propane? But at the same time, the stability of the enzyme, as it had accepted these mutations and improved activity, it had been losing what little firm stability it had to begin with. In for quick, these are notoriously unstable. And this wild events, I've gone down to where if you're growing the San 37, basically hardly have any functionally for 50 left. So that's just nothing to work with. And this led us to something we had understood and people would have understood the long time ago. I'll tell you in a really simple way. Jesse Bloom did a really nice analysis of this problem a few years ago, where he argued that if you look at all the ways that you can make a mutation, even those 9,000 single mutations that you make, and you look at the distribution and the effects of those mutations on the free energy stabilization of the protein. Some of the mutations, a few, are going to make it more stable. Most of the mutations make it less stable, right? The mutations are deleterious. And so if you're starting with a marginally stable protein, and it has to have some critical threshold in order to be functional, yet it's so you wouldn't be doing it, then most of the mutations that you can make are going to push it over the threshold and you're not even getting a protein to begin with. So what's the solution? The simple solution is just to move this distribution over. It stabilizes the darn thing so that this whole block of mutations that was formerly inaccessible, because it would just knock the protein out and become inaccessible, right? So more stable proteins are more evolvable. They can accept more mutations and they have more opportunities to acquire the new functions. This really works. If you go and stabilize that one that was giving us all the difficulties, 35 to 11, so you go add a couple of stable mutations to that. We had some in our pocket which you can find in our Directed Evolution. Then it was very easy to move forward so that with a few more generations in Directed Evolution and now all hell broke loose because people got one entire brand new genesis and started trying every method that you can think of, identifying mutations in the active side, saturating those residues, and so there was a lot of non-random stuff going on that I won't make the story confusing, but they were all basically single germicense institutions able to increase the total turnover up to about 20,000 now. That would be like the thing about another aspect of this enzyme which is that the hindermain is but a small piece of it. The hindermain which I showed during the rain is attached to this big reductase domain which has a lot of work to do because it's got to deliver electrons one by one to the heme through a nice shuttle of cofactors. In fact, I mean this is really fun machine to study. F and N actually physically moves down to deliver the electrons to the heme. The bottom line though as I don't arrive with the catalytic cycle as fascinating as it is is that when you start adding one million substrates to this enzyme or you make a new axis of institutions, this wonderfully fine-tuning electron delivery mechanism becomes uncoupled so that it becomes a very good NADPH oxbeys. We can just chew up your expensive NADPH without any product formation. So they become uncoupled to one another. And of course then highly active oxygen species get made at the heme and you get hand destruction after a few turnovers. So in fact, if you screen for total turnover number, you're trying to hold on to the coupling that was one reason that we chose total turnover number as our screening criterion. But if we haven't got this whole thing tuned and we've only been mutating the heme domain, then we're never going to get increases in total turnover. One thing I will note though as just by screening in the heme domain and screening for total turnover it caused the coupling to improve on propane from essentially very small numbers in the first propane hydroxylase all the way up to 90%. So we didn't screen directly for coupling but it came along as a result of having a highly efficient catalyst. But really I'd want to go and push it all the way to the limit. Can we make a P450? Not that it's good in propane hydroxylase but that as is good on propane as the wild type enzyme is on its preferred fatty acids in some states. So fully coupled, essentially 100%, 98% and very high total turnover numbers. So the argument that in fact we probably should be evolving the rest of the machine. It's a highly modular system. So obviously we were able to go a long ways just evolving this one part but it's also a coupled machine. So while the evolution in the heme domain was going on we were doing parallel evolution we did parallel evolution on the epimene and epidemine regions in the protein using improved team domains at the same time and then every result of doing these parallel evolutions and then re-combining the optimized domains was a protein called P450 PMO. Rudy Poisson did this work over the last couple of years. P450 PMO is a beauty, good propane hydroxylase. It has as many turnovers as you can measure in a small assay and it goes for a much longer than 45,000 in a whole cell system. It's a initial rate of reaction, 300 per minute is as good as any alkane hydroxylase out there and it is 98% coupled with cofactor consumption to product formation. So we've got a further twofold increase from the best heme domain to P450 PMO. And it's interesting to compare the rates of the catalytic rates, this ends on to the first propane hydroxylase. Remember, wildcat has no measurable activity in propane so we have to look at one of the evolutionary intermediates. K-pattern or K-advers, 8,000 fold improved KM has been reduced to 180 fold and in fact is comparable to the KM of wildcat EM3 or more like one of its components of the state so that's about 300 micromolar. K-pattern is also comparable. So it's a, for P450 it's a good P450 and just to show those numbers again, for EM3 it's very good on low rate but not manageable on propane. PMO, very good on propane and in fact has lost its activity on low rate. So now there's been almost a 10 to the 10th fold change in the specificity of this enzyme over this process. Great thing about the Michelangeloid, you've got the whole fossil record. So I've got all these intermediates in the refrigerator and if I've got somebody who's gone to the where with all, they'll go back and look at what happened during this process so now I get to share that story with you and this has someone get impolished. Rudy Pozzan took the wildcat enzyme and looked at its activity on alkanes of everything from that thing to C10 and showed that, and here what I'm doing is I'm normalizing the activity with respect to the alkane on which it has the highest activity. So 100% just means that of all the alkanes EM3 likes decane the most and then it has very, very low activity, no measurable activity on propane for example, very low on alkane but that's where we started the process. And as you go to the first alkane hydroxylase, the 1393, you can see that it now has preference for the medium chain alkane little less for decane and just barely measurable on C3. And then as you go further in the process, you can see what's happening as you go and accumulate these mutations it's broadening out, this is the one that's very unstable but it accepts just about everything and then accumulating more mutations that is improving the total turnover of the number on propane is starting to push the specificity over to the small alkanes so that right now at 11.3 there's no longer any measurable activity on decane. And then further going on, you can see that it's pushing it down, pushing it down such that in P450 PMO, this enzyme is now essentially re-specialized. It has no measurable activity of lowering, no fatty acid but it even has no measurable activity on the longer alkane. So now just by pushing on the activity of propane, it is now hardly taking to do decane, certainly not pentane on a first society has no activity on that name. So it's gone through this series of promiscuous energy and things that have much broader specificity than the wild type and then gone to propane. And furthermore, this was all done under positive selection on wrong. I think this is an important principle. A lot of people think that you need to have negative selection on top of positive selection in order to re-specialize something. But this shows that if you don't wait long enough, that's really true. But if you keep pushing it, at least with the P450 to have activity of propane, that specialization came along as accompanying the process. So I might enjoy seeing further mutations line. There's 23 total mutations. If you are 50, 20 are in the gene domain, the PMO 20 are in the gene domain, they're distributed a lot throughout the protein. There's a lot in the active site because we started getting inpatient and pushing mutations into the active site. So a lot of these were in the, out here came in the early generations. We know that they're functional because when they remove, they remove the activity towards propane. But we really have very little idea of what the outside mutations are doing. But I'll show you this cool little movie that Tom was made from the crystal structures of being free with its substrate bound, which is a fatty acid substrate, substrate bound, and then in the open and conformation with no substrate. And as I say, this is just the cartoon with the crystal structures of these two confirmations. But you can see, there's a significant conformation of genes in order to find the substrate and get rid of water from the active site. A lot of those mutations that I showed you were sitting in this FG healing region that has to close down the open thing. So there's quite a bit of, I mean, it's very important what the sequence of this region is. Now, this shouldn't scare anybody and he wouldn't be designers because these are the very good demonstration of detailed map and we don't understand the details. We would have very difficult time identifying which mutations here would affect this in the way that we're allowed to accept protein. Also, here's some interesting analysis. Comparing wild type, we do have a crystal structure of one of the intermediates, Tom Lewis's lab did the crystal structure of 1393. And that has almost half of the mutations in PMO. So we're quite a bit along the other visionary line. From that crystal structure, we can make a good model of people 50 PMO. And I'll just show you the change in the volume of the sun's very binding site as we go from wild type towards PMO. Let's get, this is a model but we think it's a pretty good one. As the promiscuity is, it comes more periscuous. We see maybe a slight increase in the volume of the site but a distinct compartmentalization and compaction of that. In fact, it's quite interesting. There's a glutamic acid that's placed in here along with the phenylalanine that seems to cut off part of the binding pocket. Remember, we're going after propane now. So it seems to have come up with a nice solution of actually dividing the binding pocket in two so that it has become significantly smaller. All right, so what have we done? We've now invaded the functional territory that was previously plot to belong only to members of this MMO-like family of protein monoxygenases. It's clear that a P450 does this and does it very well. At least the initial weights are fully comparable to natural insights working on these small substrates. Furthermore, along with this activity on propane, comes a little communist activity on ethane. So we can start this story all over again. 10 years isn't long enough but for the catalyst in the audience here, I've got a catalyst that does the direct air oxidation of ethane ethanol. 2700 turnover numbers. Air, original ground. This is the best ethane hydroxylation catalyst that anyone has ever reported. And I couldn't get it published. Everybody said, everybody knows that enzymes can do that. That's not that interesting. So it's not fair. This is a really good ethane hydroxylase. But, and it's fully good enough to get it to do it directly in evolution, directly on ethane. So the next story that I'm going to tell you next year, if I come back, would be how you can make this ethane hydroxylase just as good on ethane as the wild pet is on, or as its parent is on probate. And you have to do that by actually not putting it under there is no ethane surrogate. You actually have the screen for ethanol actually. And that's really hard in the fermentation process, let me tell you. But you can do that directly in these two little cynics 96-well reactors and we've determined some nice color ventric acids for ethanol. So that really works. But I will tell you, we're well on the way to ethane because if you look at halo methanes, this enzyme PMO has activity on all three of these halo methanes, including iogamethane whose CH1 strength is now 103. No one knew that P450 could do this. 90 of an MMO has been reported to do this reaction. So that's actually a new specificity, both for MMOs and P450s. And we're really pushing the CH1 strength significantly up. So my guess is it can do methane pretty perfectly well, but it's just finding right, binding pocket configurations of all methane in there. So that's the open question working out. Let me finish by going in totally the opposite direction. Tomorrow I'm gonna tell you a lot about larger drug like substrates because this is really interesting. This is where the, I guess where the money is in P450 chemistry because humans have P450 that are involved in drug interactions and drug metabolism and it would be really nice to have P450s that would mimic human metabolism. Remember this enzyme as it went to chemo went through a series of amnesiacs and amnias. Well, turns out if you take one of those comiscuous intermediates and you start screening it for activity on different compounds or screening mutants of that enzyme for actually a different compound, you can find all sorts of interesting activities. And I'm just gonna tell you to be right to stop. In a collaboration with Chi-Way-Wong's group and we'll reemerge its groups, we've been looking at the selective demethylation of permethylated sugars. That is fully, fully protected sugars to see if we can selectively deprotect at different positions along with Chi-Way-Wong. If you can do that with P450s, this is a demethylation like the diamethyl ether demethylation. It makes them out of a high, you can screen it color-metricly so you can add these permethylated sugars to these P450 reactions. You see nice purple color. It's active on that. You can run it through the GC and see what the region of selectivity is. If you can do that, then you can do further chemistry. You can make deoxy sugars. You could do glycosylation, and this is a single step reaction. Really, really nice. Well, let me tell you, I think it works extremely well. This is just one example for your enjoyment. This is one to get unpolished work. You take permethylated benzoyl galactose and run it across just a select panel of P450s. You can find ones that give quantitative conversion at every single one of the positions. And here's just one of the examples. These are selected, perfectly selected for demethylation at the fourth position of this substrate. And in fact, in the right conditions, you can make milligram quantities easily of this with significantly high yields. So this opens up a whole set of possibilities for new sugar synthesis. Makes it really easy. Human metabolites are interesting. So we published this a couple of years ago already. We can take the mutants of these enzymes that take substrates significantly different from cocaine and make the authentic human metabolites. So here are six hydroxy buscarones. This is a marketed drug for anxiety and depression. And what's interesting is that the human metabolite is the biologically active one. It's more active than the drug itself. So it would be really nice to be able to have the human metabolite, especially in new drugs, drug leads. So we showed that we could easily find a unit that made exactly the right stereoselectivity, the right product, right regio-selectivity, and had a decent conversion without any optimization. EMI literally was so excited about these results that they came to us with three of their drugs and said, we know that these have something like 50 or 39 human metabolites. We know what they are. Line it across your panels and tell us what you have. We looked at Barapamil, Estemizol, and one of their unmarketed drug compounds. And within a week, we were able to deliver them more than 30 authentic human metabolites, as well as some inauthentic metabolites, which perhaps even more interesting. Each one of these costs about $50,000 to synthesize a milligram of quantities. And these enzymes made all of these, including these non-op metabolites, not made in the microsomal system and stored with cloned human enzymes. Didn't make a few of them, we missed them because we didn't look very far. And some of these enzymes are very selective, allowing easy feel up to make individual metabolites. So one of the enzymes just selected this amount of group from all the other ones, got five other groups and selected this one. So 94 selectivity and could easily make 10 milligrams. That was so exciting that they begged us to start a company. I'm not the least been interested in starting a company at this point. And so I licensed them to Codexis. This is a great supplier of enzymes to, in synthesis. And they're now marketing from Caltech, this Microsoft platform for these things. So it's actually a really, it's, I'm not just mouthing this. The, these are really useful, really useful catalysts that are now available for these kinds of synthesis. So with that, I'm gonna thank two of the people who really contributed recently to this work and are starting out and did all of the catalog work and really designed these amazing settings so that people are 50 BMO and then a few other people in the lab and out at this point, thank you very much for having me here. Have you tried evolving the enzyme in the presence of a benign small molecule which might take up, which might split the binding up? A non-small one.  I'm sorry, I've never done so. Sorry, have you tried evolving your enzyme in the presence of a benign small molecule? That doesn't get hydroxidated but maybe take up part of the binding package, so. Oh, well, I'm evolving this inside of a cell so there's only, you know, 50,000 small molecules and they're probably sitting in the active site and doing all sorts of things. Yeah, but I haven't added anything specific with that hypothesis. But yeah, I wouldn't be at all surprised if there's, you know, big sitting in there. I wonder if you took a series of three steps first of all, to the T, then bringing in the given stability with some end of the first floor. And then, do you have a lot of potential and a way to do it? If you're thinking of different strategies to where you sort of tackle the stability first and you think you would have gotten the same result. So that's a, the general question is if you do it again or you take a different walk will you get the same result? I'm sure if I even used the same select of pressures I wouldn't get the same result. There are obviously many solutions to this problem. Otherwise I wouldn't have done it, right? If there was something on the solution I'm not going to use offspring. I'm doing a few thousand clones that's a very small fraction of all possibilities. I don't think that, you know, this, the one I'm trying to hear is historical and I'm quite sure that you can start and find this by a less secure trip. We're now actually doing computationally in the mining library. It's in the screen of Diagonal Believer and have plenty. They have fewer core mutations that work on Diagonal Believer. So, you know, you can do it all. You can skin that evolutionary cat in lots of ways. Yeah, evolution's great, isn't it? It's interesting that, so what you seem to be doing if people are good at this clamping down but what if you look at the size of the pocket that's the biggest? Yeah, well, it's not trying to become an MMO, right? I'd love to say that mechanisms are making a converged MMO but structurally there's no relationship. So, is the redox potential of the middle of the center or anything like that changing? We are doing those measurements now in Harry Gray's lab. I don't have the numbers. In doing evolutionary experiment in the 50s I was always very impressed by something which is periodic selection. You would have that, yeah. Periodic selection. So, I can impose selection pressures that I want but are you saying to change the selection? I would say periodic selection through the selection. So, are you changing the selection pressures as you go? When doing evolution, you get a mid-population although there is no selection pressure nothing but with you and here you've seen the old one. Well, that's kind of this idea of the promiscuity, right? Within the old one there is a new activity that appears. Remember, this is a small population limit. So, the population, we're in the limit and the whole population follows as a single population. So, I have to look at the number of functions within a single sequence. And so, those are coming up as you move along. To follow up to an earlier question when you were asked if you would do it all over again could it be done differently? Just a little bit more on that though. If you were to first start with the strategy of stabilizing the protein would that not give you a greater amount of sample space from which to begin? Is there any reason to think that given all possibilities that's the best place for which to start? I'm thinking. Probably, I was okay with wild type for at least the first couple of generations. I probably wasn't missing out on a lot of things at the tail end of the mutation distribution. Probably should have stabilized it a few generations earlier. Yeah, should have done it that way. Strangency of selection. It seems like that's going to get over how far away you've gone from wild type and then.  If it's too stringent, you won't get anything. If it's too unstringent, you'll get too many. And it's just right. So, how do I find what's just right? It's trial and error. You set up the screen and if you don't get any hits then you have to reduce the stringency. But the way that you do it is you measure what wild type can do. You measure what the parent can do. And then you just take the stringency just where parent is like 10% of what it normally was. So if you're trying to increase the stability, you take it to a temperature where parent has dropped to 10% and therefore you have this nice dynamic range of 10 to 100% where mutations can be measured. If you make it too unstringent, parent will be too close to the ceiling and you don't have enough dynamic range to the measurement. And if you take it too far, you can't measure parent anymore and you don't know whether you're five mutations away or 10, so that's how you do it. You do it by looking at what's in here. It's not black magic, it's actually really white. So there is no known natural propane amount of oxygen. And of course, since I only made 23 mutations, the music is going to be most similar to it. It's going to be similar to VM3. So it has not converged towards any other sequence that's, the closest sequence to VM3 is quite 70% identical. So it has 120 some amino acids of institutions. So it certainly hasn't gone towards anything that we know of. And in general, they don't converge to other sequences. They don't actually, because there are too many pathways to go from one sequence to the other. The probability that you'll converge on any known thing is essentially zero. And that's another thing, because what it means is there's, for any given problem, there's lots and lots of solutions. Right, they have to be. Let's do one more question and then we'll head over through your questions. So as a sort of a follow up, are the positions that you're seeing the musications in, are those the ones that tend to vary a lot between different monoxygenases? Or are they more conserved ones, or how does that relate? So you can get more information, that's a good question, that you can get more information from the positions of the mutations. Because now you really are getting the adaptive mutations. And so people have analyzed the how of these things, because they're so important in drug development. And they've identified these substrate recognition sites. So half our mutations fall in these substrate recognition sites, which count for about half the sequence, and half of them don't. Because I didn't make any additional information, tells me that we're right randomly, half the sequence. I mean, it doesn't really give us a huge clue as to where you go if you were going to design this thing. However, we have taken what we've learned about what doesn't work, right? So you can throw out all the sites that are always going to be printing. And we've taken things where we see multiple hits, and we can make design libraries from those. So if you choose five residues, then the sequence space is really very small. You can make all those substitutions at all five simultaneously, and ask you any of these, hydroxylated, dimethyl ether, and the answer is yes. So we can actually, based on what we get from random intertenancies, design libraries, computationally, design libraries, not even computationally, and start targeting mutations. So let's move all of our questions over to the computer. Thanks, guys. Thank you. Thank you.


everybody's winning this new season and they're winning the least, they're still the loser, is your argument? Yeah. Okay. Yeah. Sorry, Tyler. They have to, yeah, it's all good. I do think they have to like, I don't know, it's this weird reality. I really think we're gonna see so many one or two drop armies really on this season. I mean, well, it may be the case that we'll see a higher number because people wanna try out the sharpshooters, the Glacier Commands, et cetera. And then we start seeing a ton of one or two drops. But like that's my general expectation is that at some point, relatively soon, there's just gonna be one or two drops all over the place. But to what degree is there value? You show up at a tournament and four of your games or five of your, oh, maybe not five of your games, right? But like four of your five games, you're doing well. And mostly you're just looking at a roll-off against a lot of folks. Is that meaningful to continue doing that? But all is equal. Yeah, if let's say it's one drop, one drop and Nurgle loses that roll-off, they are against an army with a lot of bodies that has the potential to do some alpha pinning. Yeah, that could be a potential challenge. I mean, but that is why you need to have some balance in your list. You need to have gut rot, or you need to have corpus cabal, or you need to have some flies. You need to have a combined, I mean, eventually, you know, I'm obviously very biased toward combined arms lists. And that's what I played with Nurgle. And it was amazing. And it kind of took, well, anyway, it was not reflective of the norm that was out there. In the same way that Emma's list at Adapticon was not reflective of the norm, taking Rock Covenant doing incredibly well with it. And it's an incredibly good player. That's not too surprising, but when I just think about that book, my experience with it, Nurgle will find a way. It's, particularly in the hands of good players, that it's going to still be very powerful. Let me put it this way, Tyler. Like you and Emma, not dropping. I know you don't like that argument. You don't like that argument, Vince, because it's like that's not really me necessarily saying so much because skill matters a lot. Sorry, Alex. That's what I was gonna say. Yeah, like just because you're not losing percentage games doesn't mean the average isn't. Yeah, right. Exactly. Sure. Spoilers with a, with like, you know, a book that's pretty highly tooled, top players able to make a lot of hay is like not the most breaking of news, right? Yeah, I mean, I'm looking at the averages here, right? That's fair.  Yeah, like they're a shooting army where none of their shooting can really easily benefit from most of the new rules. Like they don't have much. They want to stick in sharp shooters that's of any real value, maybe thunders, maybe. They still, but they won't take that anyways. They'll want to be one drop. They can't, they don't often have like unit destroying shooting despite the fact that they're an all shooting army. A lot of the shootings actually pretty weak. They really suffer when the meta shifts to big piles of bodies. Like that is not the world they want to live in. They want to live in the world of like, you know, hey, that unit is two people and I can just focus my firepower on them with my boats. They do not want to live in the world of like, well, that's 30 dudes and that's 30 dudes and that's 30 dudes. And they're spread out over the board, like the more bigger units with more bodies, with more wounds, with more board control and unkillable utility heroes, the worse everything gets for K.O. Right? I can miss my leg. I just think they are, again, there's nothing in the rules here that says K.O. are now bad. Like kind of there is for sons. Like it's almost written in the rules like, sons, you're bad now. Sorry, just deal with it for six months, I guess, right? But K.O. it's very much the emergent property that they are just suffering from every choice. Like every consequence of what this season encourages hurts K.O. Yeah, absolutely. And they can't hold objectives with the units if they're in the boat, right? Correct. So like in order to get the GC ones, they have to hop out of the boat and be like, I got it, guys. And then just be vulnerable to any sort of ranged output.  Yep. You mentioned him earlier, but yeah, Jeremy Vesier with this new channel. He had a nice video going through some of his thoughts on what K.O. might look like. So yeah, if any K.O. players haven't seen it, definitely worth checking it out. Just, yeah, Google on YouTube, and you should be able to find it. But yeah, he had quite a few changes he was looking at with this, although it's like even thinking about like Sky Wardens, engine riggers, that sort of thing. So yeah. So yeah. No, no disagreement there. Yep, it's gonna be a rough time for our boat boys. More sadness for me. I hope their new book is way better. This is like my want-to-be army project for the year. Because I would really love to see the, I really wanna do a Lisa Votan conversion. Like I have all the stuff for it to turn it into a K.O. army. I know what all my units and conversions would be. I've like mapped it all out. I'm just trying to work on a color scheme kind of in the interim period. But, you know, like, yep, we're gonna need that book. Doug Beard, Thoughts on Beasts of Chaos. I have done Doug because the new book, we'll talk about that next week because we'll be doing the Beasts of Chaos show probably next week. The next two weeks will be beasts and then gets. So that'll be because they've announced that obviously as being going on pre-order this Saturday. So I have no thoughts yet. The cool synergies that you'll have for three days will just have to wait, right? Right, yeah, exactly. Well, ask me on Saturday when I can talk about the book and then we'll go from there. Okay. My impression, guys, is there's gonna be a lot of, like a lot of armies, I think, are in a good place. Maybe that shouldn't be too surprising because at the time of the season, we had a lot of armies that were in a good place. Most armies were in a good place, but, you know, fire slayers. Just looking at the list here. Fire slayers, Aideneth, Lumineth, probably Seraphon. Obviously they got a new book coming out, but Seraphon always seems to find a way regardless of any point increases that they get. Zinch is still gonna be really strong. I think Sylvaneth are looking quite intriguing. I know, Alex, you're getting into Sylvaneth. Yeah, I've been playing them for a little bit, but I've been playing them a little bit more now. Like, I like what they bring a lot. They can teleport, they can do a lot of these tricks, and then they don't crumple them on the list. I almost put them on the loser list. Did you? I almost. I don't know.  I don't want it. In the end, I just didn't believe in it enough. I was just trolling Tyler with that choice, so. Yeah. I agree with you. I don't think they're in a bad position. No. Ogres, Ogres is gonna be good to think in a number of different ways. Do you guys think we're gonna see or there's more value this season with Foot Ogres? I think so. Yeah, I think so. Definitely. I mean, the new book was very tilted towards Godbusters already, and this season just said, hey, let's go, right? So. Also, the double name on the Ogre Tyrant for like the three-up save, five-up board. Think I'm pretty sturdy GC. Yeah, 100%. Open as a Rhinox, holding that little banner for the plus one to hit. We're good. Absolutely, absolutely. So yeah, I mean, Stormcast, the one, yeah. Daughters Kane always seems to find a way as well, so yeah, just a ton of armies that I think are gonna be quite competitive. They find a way with their three insanely easy battle tactics to get. Sure, yeah. Is how they find the way, yeah. I'm really hoping we see Iron Jaws with some actual art boys and brutes in list for once. That would be wonderful. Yeah, actually, I saw a big wall list played against me. That was two units of 15 brutes with the War Chanter, like four units of a cruel boy shooting bull boys. Bull boys. Yeah, Nashtooth, two War Chanters and the Swamp Collar Shaman. And that actually did an insane amount of work to my Soul Blight. Of course, that's only one game, but I was pretty impressed with 30 attacks of two damage. Especially if what you think is true is more of these T-shirts saving kind of units are going out there. Yeah, sure. They'll be quite the tool to clear with it. More attacks. Yeah, you don't need quality damage. They're great. All right, let's close it out. I kept these two in here just in case we needed to reference the Galation Command, Sharpshooters and Veterans, just in case that question came up of, hey, what exactly do those battalions contain? I left that in there, as well as then the reference to the artifacts and enhancements just in case anybody was curious. It was the Night Flyer Cloak, is what I thought of earlier, by the by. That's the one that has to be set up within one inch of a friendly battle line. I apologize, two different weird teleport things, but yeah, only the one has the one inch requirement. Let's get to summary. By the way, hey, what's up, the Diascast? We've referenced some of your thoughts earlier. And if you've watched this long, first of all, thank you. Much appreciated, hit that like button. If you haven't subscribed, already do so. We're in the final push, everybody. We're almost at 100K, we might get there after almost darn near 10 years with this channel. We might actually get there, slow and steady. This is the, thank you for watching the Turtle Channel, where we go slow and steady to win this race by making only the content that seems interesting to all of us and not ever trying to do anything that's actually valuable to YouTube size. So at any rate, I think the summary for me is this season promises some big changes. I think some things will remain the same. Shooting and power projection is still a very powerful strategy. If you can remove problem units, you still win, right? You may often not need to start, what's that? That's always gonna be true, yeah? It's always gonna be true. Ranged is by default powerful. That's why I invented bows. Right, yeah. And nothing in the rules has fundamentally changed that this season. Because it's like, oh no, I can't shoot these guys. I probably in most cases, wasn't gonna shoot anyways, right? And by the way, if I'm using magic, I can still just mortal wound them off the table or whatever or careen into them and eat them with some gnashing jaws or whatever, whatever, whatever. So you may not often need that sharpshooter battalion and many of the shooting armies may be incentivized to stay one drop and control the tempo of the game, which as we said earlier, may or may not work out in their favor based on the nature of the battle plans and things like that. And then Josh A. said, Vince, thanks for the show. Can you tell me where to find your arch warlock build? It's real simple, man. It's the arch warlock plus the devious adversary, command trait, and then plus, you give him flaming weapon, obviously, and he uses his own warpstone sparks to benefit his hit and wound. I mean, that's basically what it is. There's probably a magic item in there, but I don't remember what it is. You want the claw lord to follow him around as a little buddy with the rush charm and just walk up to the thing and fight the same units he's fighting to give the negative saves. Okay, that's basically the build. He's a little claw lord companion. Okay, and then powerful infantry that was kept in check by bounty hunters is back, armies that don't mind being high drops will make the best use of the new battalions and some armies are simply straight losers in this new season, suns being the prime example. All right, so that's it. I miss anything in my summary. Alex, any final thoughts you want to leave anybody with? Yes, and I'm like, I think four moon infantry is going to be huge this season, even with like the crappy save ones in fact, and the ogres, cause like they're not dealing with bounty hunters and they're pretty durable to most stuff without bounty hunters. So I think that's going to be a huge, powerful infantry will be in check. That's a perfect example. Stalkers too, from OBR. Yep. So mostly just yes ands here.  And then some armies are going to love the new battalions, but there's stuff that we're never, ever going to take a one drop anyway. Right. Yeah, I agree. Tyler? Yeah, good summary, man. Oh, not a lot. It's coming to mind to add on to it. I mean, one thing that just popped in my head, we didn't talk a whole lot about storm cast, obviously I'm very biased around, but the storm keep almost went away entirely.  I am curious to see if some people try to make storm keep work. Like, count as three durable bodies, 15 vindictors, or even sequiturs, two by 10 sequiturs. There's a number of these you can lean into, particularly with GARDIS and Hall of Nights, five up award bubble, fighting on death on a four up, like these various things that you can stack, that I think might make storm keep interesting if you've then put in some mobility pieces with it. Oh yeah, curious, just a number of lists. I think there's a lot of space for new and intriguing list design if there are enough Johnny's in the community to push things forward. So I'm really excited about that, especially, but like we talked about near the top, I think the main change with lists right off the bat is gonna be bounty hunters going away, more bodies coming back, not so much the Glacian champions. I don't think the Glacian champions is gonna have, is it really gonna move the needle a whole lot on average when it comes to list design? Do you guys agree with that or? It's icing. If you were in a position to just take advantage of it, you're gonna be happy to have it, but I'm not building for that tool. Agreed, absolutely agree. Yeah, definitely. I'll be happy to have it, yeah. And then one last thought, kind of leave on this mission, Sean, he made my favorite point of the day to me, which was the importance of tournaments having more standardization into rain placement, particularly when it comes to this season, round one battle tactics, where he was kind of making the point of the potential of there being, say, some inequity getting created if a Tio is very sloppy with how they're doing terrain placement, which then makes, I don't know, this might be a little crazy getting this level of detail, but specifically thinking about desecrate their land here, like I made that case, right, that maybe on a number of matchups, if your opponent goes first and they cancel out your ability to meaningfully get cunning maneuver, you might be down to desecrate their land. And then if you're looking at a table where there's no possibilities with desecrate their lands, that's just one thing that came to my mind that that might be something for Tios to consider. Yeah.  Terrain will still be pretty random because terrain is terrain, it's hard, you know? So it'll be what the TO has access to and they'll do the best they can do with it, right? Like, it's just, it just is what it is when it comes to that. Like I'd love to say there's some different world, but there's not. All right, cool, very good. Well, thank you everyone for watching. We certainly appreciate it. Alex, thank you so much, buddy, absolute pleasure. Thank you for having me and like thank you for taking a chance on literally a nobody to come on your show. Nonsense, Alex has an absolute pleasure. Alex's channel is linked down below, go check that out as we imagine later. So hit that up as well as all of the links and things that we shared earlier in the show during the pick of the week, you can find all those. Don't forget to hit like, subscribe, as I mentioned. We're on the journey, 200,000, I think we can get there. It's gonna be very exciting, but thank you so much for watching. If you wanna support the channel, there's a Patreon link down below, focused on hobby and taking your next step on your hobby journey and review and feedback. There's a merch store down there, you can check that out as well. I was actually looking at some new merch store options and new merch options over last weekend with Uncle Adam. So I'm probably gonna be moving that soon. So if you wanna wait on that, I understand. But we're gonna, we're gonna, very excited about some things that'll happen later in the year there. And as always, thank you so much for watching. We really appreciate it. We'll see you next Wednesday.


Hi, today we talk about media literacy. How can it support people of diverse backgrounds to have equal chances of benefiting from the media? Cidem Bosda from the University of Bremen in Germany and University of Groningen in the Netherlands presents the findings of the research project called Included, focused on media education in a secondary school in Germany. I'm Bruderik Silva, let's talk about media and communication. Cidem, welcome to our episode. Hi, hello. A bit obvious to who's listening to us, but why is this topic, why is media literacy and in particular at the topic of your research so important? Thank you. I think looking at the society that we live in today, looking at the degree of digitalization, the importance of digital media technologies in our lives, we need to pay attention to existing and emerging inequalities that interfere with this digital society. So how are the chances of different groups, different communities for being able to participate within this digital society? Actually, this is the broader question that we need to address. And more specifically, my interest is looking at marginalized youth who are living in socioeconomically disadvantaged neighborhoods and culturally diverse neighborhoods whose families might have migration background or who themselves might have migrated to a new country, looking at these young people and how they are actually navigating in the digital society, how they are developing certain strategies to deal with digital society and digital communication and what their needs are. This is important to understand so that we can move from here to helping them, to improving their skills, to understanding also their strengths, not just looking at the deficits, but also the skills and the backgrounds that they can bring into the digital society, recognizing this as a potential as well. And the second question, of course, here is looking at the schools and what schools can do for digital inclusion and how they can help these marginalized young people for becoming better included in the digital society. And that's why also the project has acronym included as a title. And this project is actually a participatory action research designed as a participatory action research. And it is supported by the Mariska Kodowska Q-reactions of the Horizon 2020 program of the EU. And it has been continuing since August 2019. And the field research took place in Bremen in a secondary school, like you mentioned, which is located in a very diverse and poorer neighborhood of Bremen. And the project is based at the University of Bremen. And that's how I also have this double affiliation. Thank you. I think this context's background on the included project is important also for the conversation ahead. So when you started this research, what were you hoping to find? What was the research gap that you were trying to fill? As I was writing the proposal for this research project, I was looking at the literature on digital inclusion and digital inequalities. And I also realized here that there is really a gap. At least at that time point, there was a bigger gap on studies related to migration, migration background, cultural diversity, and digital inequalities. Various factors such as gender, socioeconomic background, education level were studied more than factors related to migration, ethnicity, cultural background. So that was one starting point. And also, I was really interested in understanding how schools can better support the people. Because what I see, especially in the context of Germany, but also looking at many other countries, is that schools are really lagging behind in terms of supporting young people, children, for developing their digital literacy. So these two ideas came together, in a sense, within this project, trying to understand how we can bring together the needs of the young people with diverse backgrounds and socioeconomic disadvantages together with what schools can do. And also, there are not many qualitative studies on digital inequalities. There are more and more nowadays. But as I started the project, I think that was more emerging. And I designed this project as a participatory action research, because I was not just interested in finding out what the young people in these diverse neighborhoods were doing with the digital media. That was the first aim of the project. But also going beyond that, trying to develop solutions, trying to develop interventions together with the people from the field, from the community. And what I did in a concrete sense throughout this project was I started with participatory observations in the school. I visited the school several times. And then I started doing teacher interviews and focus groups with the students. And then the pandemic started. The fieldwork continued throughout the pandemic through school closures, a hybrid education, online education, back to school, back to online. So it was an adventure in that sense for me as well. But I was really lucky to have teachers supporting me, supporting this project, really innovative, open-minded teachers working with me on this. So it ended up being a bit of a different project throughout the pandemic. But also, again, looking at the digital inequalities throughout the pandemic, which was a central topic, trying to understand how also these young people who are lacking material resources, partly, trying to adapt to online education, for example. Throughout this field research, with the analysis of the materials, we identified together with a teacher from the school, we identified topics that could be interested for teaching scenarios for media literacy education that could also help the students to develop their skills further and to bring the topic of digital media into the school curriculum. This whole field work took over a year, I think, around 15 months in 2019 and in 2020. And in the second half of the project, we developed selected, we selected certain topics and we developed teaching content and applied them in the school context, in one classroom in the eighth grade with young people who are 15 years old. And this particular article that we are discussing today that was published in media and communication, that also deals with one topic selected from these teaching modules as well as a case study. Of course, a good background. And with the pandemic, complexifying the study, let us know about the top findings of your research. Having mentioned that this article that we are discussing focuses on one subtopic within the whole project. I should also mention that the project included looks at several dimensions. For example, the general embedding of digital media in the everyday lives of young people, its importance in their leisure time, its importance in their studies, but also role of schools in integrating digital media education in the curriculum, teachers' perceptions of young people's media use. So these are other topics that the project is dealing with. But in this particular article, I looked at the topic of influencers, social media influencers, which came out to be a very central topic for the young people that I was doing the focus groups with. So over and over again, they were coming back to this topic, discussing what different influencers were posting on Instagram, on TikTok, for example, what they were going through, discussing their lifestyles, discussing their life stories, for example, in relation to several topics, using words that were used by these influencers and so on and so forth. So having seen this, I suggested to the teacher that we were developing the teaching content with that we take this as a central topic for one of the teaching modules. And I think that influencers can be a central topic for discussing, for example, the political economy of social media, the algorithmic curation, the power relations, the representations and also the cultural diversity because looking at the influencers that the different students in the school were mentioning, you could already see that they were really following closely the influencers who had similar cultural backgrounds. For example, the German Turkish youngsters, they were mentioning this very famous influencer Frant Elvito, among other influencers as a very important figure in their lives. And it was interesting to see, they also chose him as part of their assignment. So what we did as we were developing the teaching module was, we wanted to design it in a bottom-up manner so that this diversity could be included in the class discussions. And we wanted to give room to the students for explaining why they find influencers so interesting, what they find very interesting here. And we gave them the task of doing a presentation using also the iPads that were distributed through the pandemic. So that's another chapter within the research project. I will not go into the detail of, but we also wanted them to use these devices, these new devices for developing their skills further, but also at the same time, they were asked to present their favorite influencers, showing us why these people are important, what they find interesting about them. And we gave them some guiding questions and they were free to choose the influencer that they were interested in. This already created interesting discussions about who an influencer is. That was not part of their jargon, for example, that they were not using the word influencer. So it was already this discussion about what we were trying to make them, what we are trying to do with this assignment was already very interesting and showed that we use different vocabulary with these young people. And also the teachers did not know. And the teachers partly did not know who the influencers were. I think this is also a matter of age partly, but also this Frat Elvito, for example, with German-Turkish background. He is very well known in certain communities, but I think also not very well known when you look at the mainstream media scape in Germany. So it's an important alternative in that sense for these young people as an alternative representation of identity, for example, alternative representations about gender roles, cultural background, family relationships. And these were all discussions that went on throughout the focus groups. So what we did is take this topic and do these presentations. And based on these presentations in the classroom, we opened the space for discussions about influencers, about social media, a critical discussion could take place. What maybe did not, what could take place a bit more was really discussing why they chose these particular influencers over the others. That was one thing that we didn't deepen. But I think this also again links to a broader topic of how cultural diversity is generally addressed within the school context. So the teachers and also generally the schools mostly go either to cultural blindness, not discussing the topic at all, or sometimes over-culturalizing the topic, thinking that or arguing that everything is related to culture. It's very difficult to find a position that is in between. And I think although the schools, especially in the European context, but in many other countries are so diverse. We can say that they are very, they are super diverse. They are super diverse, especially in urban context. We still do not know how we should handle this situation in the school context, in the education field. And the teachers often feel that they don't have tools, pedagogical tools, and also methodological tools to deal with this. So they tend to be careful about this. In this context, we were also limited by the pandemic, the online education, of course, but at least we could show the diversity of the media content that the young people were using. There is a linguistic diversity. There is a cultural diversity. And this was very much represented in their assignments. And I think adopting a bottom-up approach like this, a project-based approach to media literacy education really enables the bringing in of this diversity by the students. It allows them to also bring in their own life growth, their own media patterns into the school context and opening up a discussion about this. Of course, there is an interesting approach to media literacy. And you indicated several times that schools, so the system was lagging in terms of keeping up with media literacy for your young students. And you started to touch this topic that I would like for you to talk a bit. Can you let us know about the impact of this research, of these findings in society? For example, in terms of public policy or educational policy, can you go a little bit further on that? So this can be seen as one study, one pilot study for really trying to understand the perspectives of the young people and designing media education starting from their perspectives in a bottom-up manner. I think this is a more effective approach than identifying important topics that should be relevant for the young people and trying to, in a sense, teach them in a top-down manner. So more important is to make them to take the topics that are central for them and try to encourage them to critically deal with this. Coming back to the influencer topic, for example, we had discussions about the economic background of the influencer market, for example. We had discussions about what they represent there and how they also profit from sponsorship relationships and so on and so forth. So it is not about just bringing in their perspectives but also encouraging them to develop these critical skills because media literacy, education should not just be about learning certain technical skills. It should go beyond that. It should make students question the media environments that they live in and their position within it and their own media consumption habits, for example. I think generally, more needs to be, like this research shows, like many other researchers, more needs to be done for integrating the topic of digital media into the school context. Germany might be also a bit of a special case here because the integration of digital media education started a bit later or now there is much more support, many more projects supporting this but it is a bit more lagging behind. I think there is also a recognition at the policy level about this but also other countries that we observe, like I think there is a gap between the lifers of the students and what is happening at the school and this gap is growing as schools are lagging behind and technologies are advancing and becoming even more central. And last but not least, I think, again, coming back to the topic of cultural diversity, looking at what these young people were telling me in the focus groups, I can't tell, I can't say that. There is really a complex linguistic and cultural diversity in their media reporters. It's such a multi-layered system and they are very skilled in terms of navigating between different media systems, between different media outlets. They have this knowledge of comparing different systems, for example. Also using media in multiple languages within a few minutes, just changing from one medium to another, one language to another, which should be recognized actually within the whole school system but also generally. Like it is not just, even when we look at these people who are disadvantaged in a sense through their migration background, through their socioeconomic background, they still have the skills to deal with the digital society in very efficient ways. And there is even something that we can learn from this. We need to recognize these skills as well, not just look at what they are lacking, what they are needing, but also looking at this and understand, we need to understand how we can also make use of this, how we can support them in these skills as well. Great. Let's focus on the now what. And you have given us a couple of hints for the future. So can you indicate to the researchers out there what comes next when it comes to studying this topic? More qualitative studies, as you said before, for example, or different methodologies other than focus groups, or as you said now in the last question, different comparisons between countries. So what's now? What next? I think this research focusing on one school could be broadened indeed, like by comparative studies, like you already mentioned, comparing different neighborhoods, comparing different cities, comparing different countries. I think these could be ways of looking at diversities within this field as well, and trying to understand what are here the patterns, what are the commonalities, and what are the diverging patterns here that needs to be taken into account. But also generally, adopting also more innovative methods, also engaged methods like participatory action research, for example, we can learn a lot from trying things together, co-developing projects, interventions, together with the people from the field. I really benefited from adapting such approach, and I think future studies can do this even more for informing policies, for informing educational curricular development, for example. This can be some of the things that we can take away from this project. Of course, Cidem, you have studied this topic before, so can you provide some additional resources about the topic that was discussed today? Articles, books, podcasts, videos, so any material that you find relevant to explore this topic further? I think it's a very rich field indeed, but I just made a few selections of articles for this. One is one article that I worked together with Kuhn Lers and Anna-Marie Anak, and we indeed tried to capture this idea of dealing with media education from a more inclusive perspective, bringing together issues related to diversity, cultural diversity, with concerns related to media education. And that paper is called Inclusive Media Education for Diverse Societies, was published in Oxford Research and Scalabadia of Communication. That could be interesting for following up on this debate for inclusive media education, and we also published this special issue, so looking at other articles within this issue, special issue where my article was placed in, that is also something that we co-edited together with Anna-Marie and Kuhn, because we care about this topic and we want to continue this discussion. And I think this compilation of various case studies could be an interesting point to start, but I also want to give a further reference to another study for people who are interested in ethnographic studies, for example, in schools and on young people, or for people who are interested in the role of schools for digital age and supporting young people. That is the book, The Class, Living and Learning in Digital Age, by Sonja Livingston and Julian Sefton-Green. I think it's generally a really good read, but also very insightful on the school context and lives of young people and how they relate to each other and how they also sometimes miss each other, in a sense, because the school is not always successful in terms of addressing what young people are going through. So I think this is also a very good book in that regard for understanding schools in the digital age. Perfect. Shidem, if there is anything that you want our audience to remember about this talk, so the punchline of this conversation, the simplest way you could provide it to us, what would it be? Tough job, but I will try. So I think important is that we need, we continue this conversation about schools, understanding the life roles of young people in terms of what they do with digital media, what is important in relation to the digital media, because otherwise the gap between the students, young people's life roles, and what is going on in the school is going to grow and grow. There is going to be even more disconnection between the two roles. And also same is true for cultural diversity. We are living in very diverse cities, very diverse neighborhoods. Schools are becoming increasingly diverse, but we just don't yet know how to deal with this, how to integrate this in the school curriculum. This isn't a general thing that we need to consider, but also in relation to media education, we need to keep this in mind. How do we address diversity within media education, the diversity of media repertoires of these young people? It was a pleasure, Shidem, thank you. Oh, sorry, that's okay. I'll, for Andy who was editing this before the punchline by Chidem. Thank you, Chidem. It was a pleasure. Yeah, this was also my pleasure, thank you. This podcast, sorry, again, Andy. This podcast is powered by Koji Tatyuprez. You can listen to this episode on the Let's Talk About Media and Communication website on Koji Tatyuprez's YouTube channel and whatever you get your podcast. See you in our next episode.