 Questions? I had a question for Eureka. So I was thinking about the lecture yesterday, I believe, on plant, you know, plant natural products. And so thinking along those same lines, I was wondering if, in your case, you have operons, but there's also the possibility for specialization, right, so that you could have orphan genes outside of the operons. But I was wondering if, maybe more generally the question is how you choose, since you have so many candidate operons to work from, how do you choose which ones you want to wake up? Is it based on novelty, or because you know that there is not less of a possibility for this kind of specialization? So prioritizing these gene classes, which one do you want to wake up? So it depends on what you want. For us, it could be something that's completely normal if you want a diverse chemical structure, but then we can deduce from the gene sequences the kind of class of natural products it will be. So we can go for something that's completely different, but also sometimes if you want different but similar activity, let's say, okay, we've got penicillin, but we want a different variety of penicillin, modified penicillin, then you can choose those that are similar as well too. And in most cases in microbes, it is cluster. These classes are usually cluster, unless if you've got like one enzyme set or something like this for natural products. The modifying genes can be single enzyme somewhere else sometimes, but it's very rare. So most of the time in microbes it's all in cluster. This is the difference from plants, a big difference from plants. But the priority, yes, well for us it depends, like I said, on what you'd like to do in the end. Does that answer your question? So I have a question for you Eric, which is how do you deal with best normal cases? That is cases where you know more or less what the actors are in the metabolic pathway leading to various compounds such as antibiotics. Can you apply some metabolic computational analysis? Has it been done successfully? Is it interesting from the point of view of setting up modifications, alterations to the pathway? What's the practice? So you're talking about the whole cell metabolic pathway, metabolic modeling? Yes, but that would focus on the pathways of interest for the production of various compounds such as antibiotics. So you're just thinking of the biosynthesis pathways for the antibody. Is there metabolic engineering going on there? Or modeling, rather modeling? Yes, because there are some modifications and stuff, so it's not directly easy. Well, I don't know if it's not as easy as a normal primary metabolism modeling. I think it's just as difficult in fact, but not a lot of people have done this. But having said that, the companies who actually produce compounds as a product, they know a lot about these enzyme pathways. They know which ones are limiting, which ones are not limiting, and they go off and change them. So in a way, they are modeling these pathways as well to understand which enzymes they need to go up, down, for example, or cofactors as well as a big thing. And also the starting material, substrates. If you don't have enough substrates, that's of course a problem as well. So it's not published as modeling these pathways per se, but industry has lots of background knowledge, I think, for modeling. But I think it's also interesting to do too. I think that refactoring of this metabolic operand can be very challenging. I'm sure you're aware of the work of Chris Voigt, for instance. So what's your take on that? Do you think that there could be also alternative approaches to be refactoring? Like, for instance, adopt things, sigma factors from the host where it comes from, or different things like that? Absolutely. So I think it was really unforeseen. Chris did a great work. I don't have anything against it because it's working well. It's just that he chose a pathway that was primary metabolism pathway really associated with nitrogen fixation. So if he had chosen a different pathway, a little bit more simplistic pathway, perhaps he would have gotten a better result. So I'm alluding to the fact that Chris's group did lots of refactoring. Then in the end, their production of the fix of the nitrogen was the same as well after they refactored it. So perhaps if he used a different pathway, maybe it would have been better perhaps. I think, yeah, your idea is great. Why not? You can do lots of other things, put different things into refactor pathways. Sure. So I think, yes, yes, I think I hope I'm wrong. So you can do lots of things. You can, like I said, with a refactoring, promoters are important. My own binding sites are important. Both strengths are important. Terminators are important. Directions of the genes are important. All of these things you have to think about and just have to assimilate it and try it. So this is for a general question, not only for you, but maybe for the whole audience. So I just wonder about the long-term stability of the constructs that we make in the laboratory. So when you want a population to obey your orders, you have basically two strategies. One of them is to enslave the population and then to punish all those that do wrong. Or you can try to make your employees happy. And then they work happily and then they may be working for you for much longer. So I think that the only strategy they have been using from the beginning is to punish bacteria that do wrong, that do wrong to your orders. But I just wonder what you can think of some type of increasing bacterial happiness by the time that we engineer them to do something. You know, microbiologists are paranoid about bacteria's stress. But I'm interested in bacterial happiness. So I don't know whether people have figured out ways of making bacteria happy at the same time that they are in a restructure. Because maybe if we do that, the long-term behavior would be more stable and more predictable. So what's happy for the bacteria? Happiness for a bacterium is easy. It's growing. No, it's not. That's the only thing that they understand. You have this phenomenon called caloric restriction. I mean, if you grow fast and get fat, it's not a good idea. So you have to find the right thing. I have a human species that is proving you wrong, right? Yeah, I just wanted to follow up on Victor's suggestion and fairy tale about microbial happiness and so on. In terms of, it depends whether your bioprocess is going to be static. You know, you do fat batch and then you use it for making something. Or you are going to have proliferation along the way, very different things. So in terms of, in Darwinian terms, living more progeny is happiness. No matter what it takes, you know, resisting radiation, storing fat at some point or phosphorus for using it. There are many, many algorithmic ways of representing it, but living more progeny is okay. And I think at this current stage of understanding life, we are absolutely, it's beyond our understanding to do evolutionary design. Not only could we construct something that would, even that is a true achievement, to have something like you describe, then having the proper maximizing the formation of some secondary metabolites by fine tuning, shine dalgona and so on. But this will be for just, let's say, a few generations. Now if you grow a culture like that, the first thing it will do is get rid of this viola, viola saying of our liking. You see what I mean? So evolutionary design, we have not even started to think, even to pose, to make the statements, the specifications about it. There are only empirical things, and I think the main concern about this is how to prevent dissemination. So there is some efforts ongoing in several labs because that's the least that we can promise to the public, not having our Frankenstein viola saying making bugs spreading in Amazonia or wherever. But this is only the beginning, and I think it is, you know, since we are gathered regularly now, I think that this evolutionary design part should become a systematic session, systematically unforced session together or aside biobricks and things which have far less importance for the future. That's the subject. Can I just... Yes, please. You first, though. Sorry. I just wanted to ask you, to proliferate is the happiness for the bug, right? Not me. Charles Darwin said it. But then they also die. So does that mean you want long-lived bacteria or you just want numbers? What do you think? It happens, you know, I know species of jellyfish, you know, the males just burst out in superb cells, but they are happy doing this because the next generation, they will burst even better but the number of jellyfish increases. So it depends exactly. You could say the figure of Mary that I want to improve is this, and then algorithmically you could try to figure out how to increase that particular trait. But we must keep in mind that it is the very capability of generating more progeny that the species of the creature that we are evolving or engineering are after. But they can die. They can die if you have lots of babies inside. It's a mountaineering. They must not all die, of course. One comment or maybe two comments to the happiness of bacteria. I think at the end, bacteria always find strategies somehow to maintain the species, that means somehow to the ones. And this means sometimes faster growth or sometimes better stress adaptation depends on the situation. At one point what we observe very often is that they form under certain conditions many, many different subpopulations. And when I look for all these modeling studies, I mean how we ever want to integrate these subpopulations is another challenge which is of course almost underestimated. I mean you can just knock out one gene and suddenly find heterogeneous behavior or you can overproduce and find homogenous behavior. So you can never predict. Any comment? On your side? I'll take a different stance. I think you can predict to some extent because you can analyze the different components that you're engineering into things like bacteria and work out what the relative fitness decrease of running that component will be for the cells in the conditions you want to grow. You can analyze DNA sequence and look at the predictability from the sequence level of there. That sequence being deleted or changed due to things such as repeat sequences. Maybe for example the way you've closed the plasma leaves a similar sequence multiple times and that then is going to have a more likely chance of being ejected out later in design. So although I don't think we can easily work out ways to keep the growth rate perfect to avoid our engineered bugs from being outcompeted by ones that have not been engineered or that have got some mutation. We can do things about the design that can improve our chances and our design. Our DNA will be kept for longer periods of time because certain components cost the cell less effort so RNA based regulation probably less cost for the cell than transcription factor based regulation and designs that avoid certain sequences where transposons are easily going to come in and cause damage to your DNA and also be avoided. So you can do something about it but ultimately I don't think we really want to get to the point where they grow even better than wild-type E. coli because for example if you're doing this work in E. coli that would be breaking what was set out at a cellar mark in that our lab work in E. coli would not create strains that grew faster and would fit it than the wild-type natural one. Alright, if I may introduce my own view of that I think it's a matter of domestication and styles of how we domesticate bacteria at least think of cows and maybe some wheat and corn. From that point of view actually there is probably an incentive if you want your bug to run during months in a 1000 cubic meter fermenter and not change properties too much to be perhaps a bit on the nice side of domestication that the cells are somewhat happy with what you added to their constituents so that they stay robust and Philly does not agree because I know the complaint of yogurt makers so they are making cubic meters of yogurt every day that God makes and sometimes it tastes like cat piss because some viruses were there that resisted so the only way they have sometimes it represents millions of euros actually a fermenter of yogurt like that is lost so they have to... they have a real incentive in understanding and steering this evolutionary thing they are absolutely no model, no way, no other thing like no more than so many variations and stuff so that's what I said or I said this thinking has not even started so the shuffleback of what Philly says and is that you know you said we have to think on evolutionary engineering design, part of the design should be... so what I want is to have things that don't evolve at all and what I want is to have systems that can be derived and then that takes me to the other side of the coin is this issue about growth so I think that what will be growing is also in engineering and so on and that's a component of the whole system that makes the whole thing very different from what an engineer has so an airbus does not grow or a radio does not grow, right? so I just wonder whether we can also... we're also thinking bacteria in terms of something that grows from the time so I would like to have what would be to something that we would call an adult bacteria so we grow at the beginning, we have a long adult life we do our jobs and everything and then we peacefully die so can we think on adult bacteria but they don't grow however they do what they ask them to do and everything so I think that it's been touched laterally this issue of uncoupling growth from activity and to this day I think that this is still a big issue how can we use synthetic biology to uncouple all together growth from a catalytic activity not stressing the cells because this is the standard way to do it and still having a happy adult catalyst I don't know, any other ideas about that? if I had them I would most probably not communicate them here rather silently pursue them I think your problem description is very accurate also the solution would be great I think nevertheless it's remaining a very tricky endeavor because these regulatory processes that define whether the cell is happy to whatever definition can not really be pinned down to a limited number of things we need to turn this up, we need to turn this down so to me they seem to be distributed over a large number of aspects and to change them will A. require better understanding and B. lots of interventions at different spots I think what you describe is great but it will take some time I can add a comment perhaps so antibiotics or secondary metabolites as you know are not produced in primary metabolites they wait until they stop or reduce growth and that's the only point when they start producing so maybe they're happy producing these compounds because they're not competing with growth so one of the ideas of pharmaceutical companies before as well was to engineer so that they only produce when they stop growth you engineer the expression so they come on only when you have a fixed mass and then you can switch it on and off whenever you want so I had a question for Pablo so one of your final results was very nice that you showed that biofilm or organisms in biofilm had higher degradation of the coral butane I was wondering if you had maybe at this early stage but any speculation at least on mechanism for this is it greater chemical resistance or? Well one of the things that for sure is different in a biofilm is the way the cells can withstand the substrate it's extremely toxic so you can only add 0.5 million more so cells in the biofilm are much more resistant to the substrate itself which is toxic than in the platonic state that's why we think we recover most of the deologinase activity in the biofilms