 I wanted to just mention, I just wanted to mention Leslie Orgel, who is a giant in this field and it's actually very, very hard to think of anything in this area, kind of, that Leslie hadn't thought about or thought of in some way or other. And I think also as the theme of this morning session, I think I would like to quote Orgel's second law, which is evolution is smarter than you are. This is really nice, okay. Well, that's one question. I had a question for Philip Mariev, an intellectually demanding question for me. How do you know which mutations are adapted or relevant when you have 6,000 mutations in your run one, 8,000 in run two? So one, heuristics, you actually, of course, in some way or suggested, is to look at the overlap. You do more experiments, look at the overlap. If you have in the end 20 mutations that are overlapping, it doesn't mean that they're sufficient. So you could clearly put them in a new strain and see how it goes, but you probably require some of the other mutations that are found within the 8,000 or 6,000. Do you have other heuristics to find which are adapted or relevant? I completely agree with you. We are so happy to see that many mutations. You know why? Because we thought of referees, if you find only 15 adapted mutations, they are going to ask you to take each of them and put that in a separate strain of 6,000. No journal ever nasty and francophobic and whatever will ever pass. You must have found ones that made some sense, though. Are there any binding proteins? Definitely. The replication apparatus is under fire. RNA polymerase is under fire. The hyper-system is mostly noble enzymes of mutic acid, but not so much a nucleotide metabolism. There is thymidine kinase also is a target of mutations. Do you think Chanel and other proteins that bind DNA? Well, some of them are, yes. But polymerase, in each light age, we find that polymerase. Now, I don't think that we are going to really study, because in a trajectory like that, you find interesting things all along the way, but our main target is to really leave the solar systems. Are there all five polymerases or just three? More than that, you have now up to 10 mutations in polymerase. In all five, in how many different polymerases? Like just pull three or one and three? No, no, several. I think it's three out of the five. The question, I think that maybe one has to go from the chemostat or better from the turpostat to the gradostat. Because I think this is clear that gradient, especially spatial gradient, some parts of it might be ice and some parts might be not ice. That means then we can coexistence via niche effects. And this creates a lot of interesting patterns. You showed that picture there, it's very familiar. There, if you have really environment involved, then you get crazy patterns. Yes, there are also unexpected ways of helping evolution. For instance, having predation. If you have predators, you have a faster, even in a completely orthogonal way. Yes, you pointed out an interesting situation where the direction of mutation went differently after you finished the initial adaptation phase. So you went A to G in one culture and B to A in the other culture. Could that be explained by the acquisition of a specific mutator mutation in one of the two lineages? Actually, we tried to see whether there was such a pattern and we did not find it. There is no clear... Because it's as a sudden, as soon as it was only accelerating and the generation time was no longer imposed. We saw this trend from the turn of the evolutionary regime. Transcription side, so you have mispairing, but it's also possible. I was curious if you saw more mutations in the transcripts, but also how you would be able to tell that it's from the RT and so on. We had mutations in the RPOS and the transcription machine or in the RNA polymerase. You don't have tata box, but clack-clack box and stuff like that. So RNA polymerase has to adapt to that, but we did not do a thorough transcription analysis along the evolutionary process. So we don't know. Was wondering if the cell remains able to execute a result program, for instance a fade or something like that? No, since they are always growing, they no longer have a stationary phase. So you can freeze and thaw so then okay. But you cannot just equalize the model organism because you can just let it on the bench for the weekend and then you come back on Monday morning and you can resume your experiment. They die very, very quickly and so everything that is not to be maintained during the evolutionary process is lost. So phages, they are less sensitive to, I think, T7 and T7 was tested and T7 seems not to infect it as well as previously. A kind of forward-lapping question about the genetic stability of these mutations. How stable they are? Well, they are as far as we can study because it's difficult to just have an isolated strain from that and study it because they die out and so on. So we have problems of reproducibility, but we don't know whether it's purely phenotyping because they die or whether it's genetic. We don't have indication that they are very, very mutated. They are steady mutator of the accumulator of the order of one mutation per cell, per generation, which is high enough because it will reach salmonella and foreseeable future and so on. But that's what we can say. Please. So I don't, maybe this is, as I understood yesterday's talk, you're looking for new pairs that are stable. That's part of the criteria you're looking for. What if you look for ones that were stable? You'd have to do a co-selection on A and B or X and Y or whatever you want to call them. But where the cross-hybridization with the naturals are not stable, you could enforce speciation. Absolutely, definitely. And that's actually one of the nice goals to consider is to try to affect speciation. Oh, exactly. Thank you. I have also a question. Sorry, Fina. Now your turn is coming up. Hi. Please. In evolutionary biology, it's very often the case that if you have these alternating environments that you get special phenotypes that allow themselves to adapt to speciation and bad hatching. Do you see that? I cannot say that we have, that we have all other media in which we could test them and so on. We saw that they were very, very unfit. And we don't have, I don't think that we can answer. You mentioned the finding became toxic. Yes. Have you considered running your experiments backwards? Yes. You see that the cock wheels are turning in the right direction in your audience when people ask this question. I don't think that we are far enough. I mean, at the beginning, you know, we would put it on the timing and see a reversion mutant pop up. They no longer pop up. So we are not far enough. But time will come and we will definitely do that. I think you just pick up too many compensatory mutations. Yeah. That's a one-way street. Yeah, definitely. A question for Phil, however. You rightly pointed out the importance of local concentration effects in these stories and scenarios. I was wondering whether it has been explored that small oligonucleotides that have the possibility to pave a surface or a volume would allow for the concentration of other or same oligonucleotides that would have some catalysis properties and would, at the same time, protect them from pollution. Yeah. I think surface effects have been explored by some people. They're simply by tethering oligonucleotides. And yes, obviously you can then go through concentration and wash cycles and, you know, you can overcome product inhibition to some extent. One of the problems of coupling things to surfaces is that at some point, you know, things need to come off the surface again. So you need to have some sort of regime which will then strip the surface so the cycle can start again. We haven't explored that, but, you know, some people like Fomki Adrovski, for example, has looked at things like that. Certainly there's also simulations which show that you need compartmentalization as a prerequisite for the convenient evolution, but under certain circumstances just local surface patchiness might be enough to isolate, for example, a replicator from parasites. So yeah, I mean, these are interesting ideas, but we have not explored it yet. Phil, I have a related question. Chesedore, very nice talk. And did you ever check whether pH changes also play an effect in these otactic phases? Yeah, yeah. Because it's known that the ice crystallization or during crystallization, certain, for example, in the lab, the buffer components will crystallize later or earlier, and then this might affect the pH in these new phases. Yeah, yeah. The pH changes by about 0.5 depending on the buffer. It changes by about 0.5 units, but when we try to compensate for that, literally nothing changes. So I think the pH change is not the dominant effect we observe. I think the dominant effects are the temperature change, the concentration, and possibly, although this is very hard to prove, the surface effects of the ice crystals, because there's a very, very large surface that is generated as the ice crystals grow. So we're just discussing what historically was the temperature situation back in the day, and also related to that, I assume most of the ice is salt water ice. So are your conditions closer to what you would have by freezing salt water ice or fresh water ice? Yeah, okay, so the first question I can't answer, neither can anybody else, because I think there's no wrong surviving from the very early days on Earth. I think their estimates kind of based on so-called CERCOM inclusions, which give you only very average temperatures, but I believe the consensus, although I'm on thin ice there because I'm not a geophysicist, I believe the consensus is that the planet acquired a hydrosphere very quickly and cooled to about a global temperature of approximately 40 degrees within the first 100 million years. So assuming that and actually the fact that the solar output was only about 70% of today, I think it's certainly not far fetched to propose ice maybe at the poles or seasonal kind of at higher elevations. I mean, I would say, but it's impossible to be totally certain about that. I think the conditions of our assays are mostly compatible with fresh water ice. And what you'd find is that the high salt concentrations that you'd find in sea ice are mostly compatible with not just nucleotide chemistry, but also assembly of membranes or generation of amino acids. So I think all of the sort of most credible scenarios for generating these things need to take place, I think, outside the ocean. I think there is some chemistry which has been proposed to occur at these various types of hot vents, but it doesn't necessarily have to have the origin of life where you need to make all the stuff. I think some of these chemistries kind of are okay as foundries sort of where you assemble all the bits that you need to build the car, but I think the car needs to be built in a more benign environment. Sir, in the beginning of your talk, you showed us a nice picture of Campbell's primordial soup. Do you have any idea how diverse this soup must be before and after so you also assemble the thing of your car? Yeah, I'm not a primordial chemist. So I think there's various people have advanced various scenarios, but there is now a number of, I think, very credible studies which yield, as I said, a number of amino acids and lipids and certainly some of the nucleotides from things like HCN and cyanogens and acetylene. So basically highly reduced components which I think you find, for example, in comets and in outer space highly abundant. So presumably given the probably almost incessant bombardment from outer space on the earlier, certainly these molecules would have been present. But this is sort of outside my expertise a little bit. I mean, what I would say is that there's credible scenarios to take you to nucleotides. Some other people, for example, Jack Schostack and David Deemer and others have shown some credible pathways to go from nucleotides to oligomers and really kind of this is where our work would start. But presumably you would, this is pre-store cycle, create different combinations of oligomers every time. So you would need sort of different amounts of diversity, I think, to start with. Yeah, I don't know actually. I mean, I think that's a very good question. What would be the minimum amount of diversity that you would need to start with through iterative recombination to build up sufficient diversity from which phenotypes could emerge? I think these are open questions. I really don't know the answer to that. Because there's a question in your abstract, you mentioned the thing that it might be an accidental freezing of the actual set of... Yeah, I think that the frozen accident refers to the chemistry, not so much to the process. I mean, I think, well, this is, I mean, again, like everything to do with the origin of life, this is controversial. But I believe that the chemistry of life is built, is basically opportunistic. So life got started with building blocks that were abundantly there, given by Earth's pre-voltaic chemistry. And once you've made that choice, it's very hard to change. As Philippe has shown, it takes a good look. Could you use this idea to get something going? A synthetic gravity, that's right. Well, I mean, I think, as I've shown, in principle, there is probably a number of backbones or polymers that are capable of heredity, sort of genetic information storage, propagation and evolution. And certainly, probably, you know, not just backbones, as we've shown yesterday, as we've seen yesterday, there are probably different, you know, base-pairing schemes to encode information as well. Let's say there is a large but finite number of ways to store and replicate information. And I think, in time, we might be able to explore it. Just to be clear about that, to my knowledge, there still only is one biopolymer that's capable of amplification of heredity. That's DNA. No. Thio-DNA, 4-prime, Thio-DNA is also able to... Okay, okay, but that's what we've been talking about. Well, RNA, there's RNA viruses, they clearly are capable of heredity and evolution. Okay, so RNA, in DNA, predominantly RNA in some cases, but just for the language to be clear, I think you should be careful about calling those genetic systems in heritability, because you still are using the DNA and the amplification. And the amplification is a huge component of that. No, no, I agree. I don't call them genetic systems, but I think heredity as a process of information storage and propagation is a nice sort of short form of kind of saying. Yeah, at some point things become short enough that people misunderstand them. Okay. X and A polymerase, X and a dependant, it's only a matter of time. Sure, but it does require... But it's not yet... What's that? They are not yet available, but there is... I will not ask the question when they are. Right. Perhaps I wanted to make a comment about spontaneous evolvability and so on. And you know, you find, you read it very often, people like to see star evolution, then chemical evolution, then biological evolution, as if it was a single process. Presumably it's not true. When heredity kicks in, you have completely different regime, including generating people who think like us and who can transform and morph completely things. So in terms of implementation, in synthetic biology, I would advocate for going as artificial and improbable and non-spontaneous as we can, and not the other way around. For setting up life, you know, start again, because actually the specification that you have to respect is enormous. You really want to close the gap between some chemicals or ribals or whatever and real life or evolving system. You have to... Not only it's a matter of chemical design, but you have to follow this, all the continuity of these logical steps, which is daunting. Now, with organic chemistry, you can simplify a lot. Because you can arrange design systems in non-accus solvents and so on. And implementation might not have all these constraints of continuity and logical design, stepwise. You can make big jumps. That's a little while in the back. And this is very fast. So you engineered equal like a new methionic metabolism and had the single growth rate after 40 days. I was just wondering if you missed that population, if you wiped it out, what would happen? Would it both be complete? Would there be two populations in the next 10 times? Actually, one of these lineages was evolved long enough in turbulence as it was done in Berlin by Hutter himself. And actually, at the end of the evolutionary process, in terms of that, he made competitions with wild type and he grew faster. But of course, it was not such a demonstrative experiment because we did not adapt wild type E. coli in the other same conditions so it should be the outcome of the same competition independently to measure. I tried to allude to that earlier with this Turing test. It is confronting adaptive challenges that we can really measure the improvement in evolvability and so on. But such experiments, this is the future. You are right. But at this stage, these machines are not too costly but we have a limited number of them. The first objective is to reach the outskirts of modern nature, ASAP, and then we play competition games and stuff but we are not yet there. And it is a matter of resource allocation and so on. So that is Turing test. Is something that Turing test has a follow-up? Yep, it's a... As I said, then you can use competitions, you know. You can adapt a Floyd's strain and put it in competition with evolvability so that the bases feel good, invade the genome for a short while, do the same with Ishiro's strain then put them in competition. At some point, you know, this kind of experiment, among the different bases that you mentioned, you could put the bases in competition for invasions if you go out, you know. The races are not only between organisms but you could use organisms to sort out the functional benefits of different bases as well. I'd like to invite some... but briefly from the audience of this idea of the Turing test, what about when we decide that synthetic biology has become a real biology or something? It is real biology. We don't need the test. Yeah, it just depends on what you're interested in. If you're interested in solving a specific question then one way to do that, I think what we're referring to here is how do you tell whether something's gotten as good as nature produced? Yes, sir. The situation reminds me very much through the problems we may have to do with designing new algorithms if you want to sell the industries. They say, we know use is better but we know ours to implement that costs a lot of money and we know what it makes wrong but we don't know what your skin makes wrong. This maybe is an obstacle. Life is algorithmic chemistry, definitely. When you talk with computer scientists about this kind, they see an organiser as a competing program. I think it's very pertinent this way of thinking. Now, you can do that in silicone, the kind of computing that you have and the evolutionary process itself as a kind of statistical programming and so on. We don't master formulae. What? You're obviously pushing very hard on this idea of completely synthetic life and building new chemistries and new algorithms and new solutions to what we have now. Why do you think society and the public would think that? In this room, we're all kind of scientists. Don't tell anyone. We're going to make life that you've never seen before and it's not even following the rules of life. Yes, you are so right. I don't know, but you know people look at domestic animals, they like them. You see these paroles, cattle, they look so happy, they are completely, they are genetic variants. We are north of them. They no longer look like rocks or stuff. We would like to make them like xenobiology creatures, like their pets. They look like some kind of monsters. But I agree with you that it would be super. We are thinking of having this big European, big European, like the brain, the graphene and say larger than life. So we are going to use everything, all competencies, all kind of different skills and so on. So as to make a second nature that would be very respectful of course of everything, gender, things and stuff. But completely artificial, so as to help the industry. But I think it's easy to convince technocrats of that, but the public is just... And I think maybe to me the most interesting thing in varying the chemistry is to understand what makes the chemistry of life special, or not. About your Turing test analogy. So you said something about once we get to a certain point we have to contain things ironclad way. But it wasn't clear to me if you select for something that has a gain of function that outperforms wild type and you've been doing that for a long time, but almost every time when you do that and then you test them under some other fitness condition it never can compete with the wild type. So is this really a problem? Yes, I think it is. You remember Jurassic Park, life finds a way, and you know, I would not bet on the fact that E. coli will never find a way to make chloro-uracid and become prototrophic again. Now if you treat with hypochlorite, cytosine or whatever, you get chloro-uracid in the juice or with some chloro-peroxidase and stuff. So I think chloro-uracid is accessible, it's within reach. So if we think along this line and we want to contain organism we will have to go a long way. It's not going to be easy and scientifically it's going to be very demanding actually to get to a stage where we can make sure that there are something like 10 to the power 30 genomes at work, at work, cellular genomes at work on Earth. Well, that's big. That's not that big. So it might be clever enough to find chemistries that the whole biosphere could not tame or master itself. I think it's a big issue to go in the chemical reprogramming of life beyond the search that the biosphere alone is able to do. But of course, I'm passing it in a bit... I know that I'm here in a mathematical institution let me know too. It's a question for mathematicians. How can we measure the search of what is searchable by the biosphere and with our little knowledge, skills how can we say at this point there will be no way of having spontaneous evolution make these Jurassic Jurassic part monsters of ours. Isn't that also not only a question but also a question of the conditions that life evolves into. It's also about what kind of business artificial conditions that life can adapt to in that survey. It didn't have the need to go there. That's right. But we should not be too optimistic on the fact that they are de-evolved so that they were just in some plumbary and that they will never re-adapt to the wild world. We don't know, we must be pessimistic about this. So I think that there is another strategy to reach a bit of a single, which is not to change the chemistry but to change something else. That's a concept of life. So can you comment on that? Do you think you can really reach the same level of proliferation? It's a serious business. When you have cattle fed with beef, you get prions in the cycle. So proliferation when you have proliferative latency, having a cycle, a nutritional cycle and so on will lead to proliferation and improvement by natural solution. We saw it in many infectious diseases, prions and so on. So whatever I think it's a matter of nutrition for containing because if it is only a matter of auto any entity that is able to take things to assemble them and proliferate if it can find them in the wild or make them through some metabolic reactions, it will invade its embryo. This is Darwinian selection. So to prevent that, even if we have different genetic codes and so on, it will not be enough. We have to prevent their material proliferation. So we have to make them absolutely dependent on nutrients that they cannot find or make. I think it's a question that is very well stated. They must not find their nutrients. Otherwise we will have prion life whatever. And we have to be super pessimistic about it. Not say life will find away. For myself, I tend to come down more on this side. I think that in relation to the question that came up yesterday, sort of what new activities looking at the case of proteins could we all I think those would be very specific activities that you chose specifically for a certain goal like a therapeutic activity or something. And I think for the notion the question as to whether or not something could grow to dominate in the wild I'm not sure that anything that at least as I look at it, there's anything out there that is going to make things grow faster or be more adaptable in general. Nitrogen fixation is a shame. It's one of my favorite stances. I show you metabolic pathways. Some of them are nice, some of them are just so I think the argument changes then. Nitrogen fixation if you make it easy and if it is made in the common genetic language it will spread out. So you'd have to have specifically the ability to fix nitrogen contained to one selectable thing. Yes, but there is such an incentive that nitrogen and so on. There are probably other ways to do. Nature has found this one. There are many chemical reactions very easy in the industry that life has not found yet. So introducing such capabilities like metathesis or stuff like that you might provide incredible incentives in terms of selection and metabolism. Okay, the last question is more a question or as a remark we have been discussing yesterday the modularity I think we have to take into account not just the chemistry we have a lot of all the modular level biomechanics and the signaling is getting more and more important we have to understand it better. Therefore I'm not against biochemistry but only in the opposite. But we should not forget. There is more in the end. Especially biomechanics and motility of species is very important about which you cannot catch with a compass. Well, when you look at a ribosome it does very fine-tuned superb chemistry but you can also see this as a mechanic robotic thing. It's a mixture. Yes, the pieces are not so well separated. It's not separated exactly I'm coming from a modeling side it's overlooked. We look too much at chemical reaction system not taking into account biomechanics which is an important feature of structure from a... Empirically making covalent bonds is what we master best as seen from very far away and you heard yesterday this talks about elaborating complicated base pairs. It is hard. So we know and engineering biomechanics at the same level and doing physics in the same way would be much more difficult I think with the current knowledge that is clear. Okay with this I'd like to thank both speakers again.