15:05:48 Ok, I thank the organizers again 15:05:50 For giving me a chance to give a second talk. 15:05:52 So this talk is on crossbreeding ecological interaction, and I should warn you this is a really a new topic for for for for our lab, we mostly look at, as you heard from last week on physiology, Michael voc theology. 15:06:23 at some simple instances of species interaction classmate interaction from the physiological perspective. Right. And so this is not be a big change of gear from France. 15:06:28 Very granted complex systems to much more simplified says, but what you will see is that even was this very simplified system. 15:06:41 It's much more complex than we were expecting. 15:06:44 And so it actually this this entire project felt very much like the first experiment to a project we ran into doing my transition from doing sort of a theory and a modeling of a biological system to doing experiments. 15:06:59 And at that time, our first project was the study of a lack the control of a laptop bro. Okay. And so the idea was so me and my first graduate students doing experimental biology, Tom Kuhlman, we're going to do two three weeks to sort of, to, to learn 15:07:18 what was already known for that system, and then go on to do something interesting. Right. But at that time that project took eventually took three years and it was his thesis. 15:07:28 And so this very much felt like the same thing. You'll see we're looking at the very simplest kind of a class Venusian thinker, and it's. 15:07:46 It took us three years to give the story. Okay. 15:07:46 Oh that's always that's always the thing you have to learn, and that's the mental block is the most difficult part. 15:07:54 Okay. 15:07:56 And a lot has to do in each of these case from the persistence of the experimenter who will not just take the commonly accepted view as it again. 15:08:05 Okay. And, Yeah, I don't know how they do it. 15:08:09 I've been there yeah so capital ominous is the, the person who spearheaded this project, and maybe it's a specific questions. 15:08:19 Alright, so the overall 15:08:26 topic is on diversity which we did quite a bit. 15:08:31 Last week already. And it was basically how in very simple experimental system how many species many means more than 10 codes. 15:08:50 So they took specimens from leaves or soil maybe, and then grow in glucose minimum media. Right, and do this across dilution cycles and end up with quite a number of a species. 15:09:08 And then later on in the program I think you're going to hear this study by a tornado barrel from Jeff was a similar type of things to sample complex environmental sample and and do these gross national cycles. 15:09:25 Right. And then you see that now they look at the diversity species richness of the function of the, the number of a carbon source of pride and even was one carbon source. 15:09:40 Right. So this seems to explicitly violates the principle of competitive exclusion. 15:09:49 And just, just to remind ourselves here is the MacArthur so consumer resource model again. And I don't need to go through the details. The, but the the is well as you know that model supports what the number of species that can be supported is less than 15:10:18 equal to the number of resources. And it was pointed out in Pankaj paper that the could one possibility of supporting many species is there's an extensive cross feeling. 15:10:31 So, the suspicious that is spread out, well here's just one, perhaps many, many metabolites, and they serve as a sort of new resources to to to support diversity of the system. 15:10:41 and at the level of equation when simply as a term about across. 15:10:44 Okay and just give them a constraint that carbon has control over something and then can see this happen system to support manage species. 15:10:54 Okay so, but then as now. 15:11:15 that whoever's succeeding. Its metabolites and needs to substantial need to secrete a substantial amount and types of the metabolites. 15:11:28 Hmm. 15:11:28 Yes, it could be like, Yeah, sure. Yeah, it. The, the show to at some level if you accept license just as a child and there's absolutely no, there's not a question not know puzzle about diversity. 15:11:39 Maybe that's what they do and what we see in this case they can do a lot better than nice. 15:11:45 Right. 15:11:46 But then, if we're under this, this type of assessment area with species we're growing and a supplement metabolite then there's really a kind of a, a physiological question What is the driving forces that enable this that allow the species to succeed 15:12:02 pressures Metallica as the cookie themself for growth. Right. This is depicting a kind of a socialistic community right where everybody's helping each other and the many, many types of growing. 15:12:18 So let's think about several cases of a metallic Crossfield that we know, and the one is the one that I actually worked on and it was a well known case of overflow metabolism. 15:12:20 So. 15:12:30 So for some when I was growing up glucose is extremely acetate, and for services exclusive ethanol, now it's a widely occurring phenomena in microbiology, And this is despite the fact that if, if, if, if, Glucose is burning to carbon dioxide can generate 15:13:04 In eroded condition. Right. And so they're in this paper with describes a reason we think what's happening that drives the call I should do this massive solution. 15:13:14 So, here, the amount is massive. It's really enough to, to support kind of a, an oldie worth of a self growth. Okay. 15:13:22 The end there's a reason and there's a reason why he has to, if it can grow fast. The rather uses less efficient enzymes. So that's the cheaper so we can allocate resources to pass Rosen with experiment to to test and establish this scenario. 15:13:39 Okay, so very special reason for doing this. And for the context of this that diversity ecology. The problem is that this is not going to, you're not going to get this system to exclude by 10s of 27 metabolite, not to mention hundreds. 15:13:53 Okay, so for a very specific reason it is doing it to to to save 15:14:08 then there's another contest where excretion is well study, right, and this question already. 15:14:09 to save the machinery. Okay, so you can measure what's excluded there's a little bit of other stuff but mostly it's just, 15:14:17 Just to your point about acetate excretion right like the. 15:14:34 special lactate and other stuff, so you can at least get to like five or six that way right. No, no, but but but usually in this in the secretion. Yeah, you may get a few right so in the case of Nicole I use them you were talking about the amount of the 15:14:36 It's, it seems to me like it's a sort of historical accident that that's the thing that Nikolai secretes right that like other species secrete other things in their overflow oh yeah yeah so it doesn't have to be 15:14:54 one that was massive isn't 15:14:54 another type were really class feeling is I was integrally involved is anaerobic digestion. 15:15:01 So, some of you were this spring. 15:15:06 Zoom forum that offer the former ran and it was very very deep literature very complicated. Right. And then, In this cases. 15:15:15 So it's anaerobic and also lacking other other 15:15:20 chapters and you you you you feed a system with Apple hydrates from the various carbon sources, right and the first have us know Genesis would be to a papaya and a whole bunch of ethical generator. 15:15:32 Right. And then these are turned into a few other substances but but the the uptake of consumption of these guys is really in another easy energetically not favorable process and for this to go. 15:15:50 You actually this is couple with Nathaniel Genesis, so that you can pull the Fluxus way and get rid of a hydrogen and eventually the idea is to produce the to discuss the escape the system. 15:16:05 And then, then you had the host. So in this case, crossbreeding is really essential, and indeed the whole system will basically from all of this stuff until you get to the endpoint. 15:16:14 If you don't get to the endpoint intermediate stuff accumulate and then stop the reaction, you have to go all the way. and the key about this process is, it is that the, they're really munching on very tiny little bit of energy. 15:16:30 Okay, so these, these, these guys really have to work together, right through these difficult reaction to get the system. Okay so overall rates and very low. 15:16:40 And what this and this really community people that been studying this for decades, right, and it is. Well, I mean it's not in the kind of the context, we're studying but then but then many specific parts of these reactions are no. 15:16:57 But, 15:16:57 but I bet this is not what's happening Josh, I think we all agree we agree on that, because this is a much more high energy, kind of a flux situation we're talking about in this at this April solutions. 15:17:12 So what I'll describe today is the beneficial row of this expansive class feeling. 15:17:22 Right. During a robot growth and English in situations that could. 15:17:27 Well, we're not we're not exactly a selling Josh experiment or materials experiment, but the type of a system that's I will say that simple. 15:17:37 And so our system is derived from auto. 15:17:45 The organizer who will come next week. 15:17:49 run a lot of ecology, from also. 15:17:53 So he has this tightening the grading system. We have a cotton cotton cotton coding and then the beats and the show this stuff into seawater right and then they collect the various time and then look at the sequence of a bacteria. 15:18:11 Curious on the system. 15:18:11 And so, in the first paper. 15:18:17 paper. 15:18:18 You see, so as a functional time various things that get to jump onto this beast, and we just look at two species from this connection. Okay. And so one of them is a reveal species 1801, and we pick it for the, for the reason not entirely. 15:18:37 That is one eight or one suffers. 15:18:47 Okay, so we study that species. 15:18:47 And then another another species we look for the species knows more than that. 15:18:54 Okay, so, so that is the two species that that gets accumulated as the one on one is, is actually a kite integrator. And it came out at first and then there's the three to five models. 15:19:11 Okay, so while we were looking at. In reality, we're looking at a number of species a medium that these two sort of looked very simple and interesting so we decided to look into them. 15:19:23 And the entire story is about this ain't gonna be okay well. 15:19:30 So, first we look at the growth of the monoculture growth net which is the model of a kite. 15:19:39 And, well actually first song titles. So one of the a gross on Titan, but be does not. 15:19:45 Okay and a also gross on the monitor, and Peter's not. 15:19:51 And so then, to do 24 hour gross dilution cycles on growth neck as the sole carbon and nitrogen source. 15:20:01 Then, we see that the quickly settle down to a ratio of about one B two to a 15:20:13 This cartoon is the lack of the shelf shrimp and just a hard substance, not dissolvable subs. 15:20:17 title. 15:20:20 It says, Okay. 15:20:24 Could you say a little bit more about a and b. So, ba, is creating these EXO these enzymes that are cutting up the Titan right and producing the monitor. 15:20:34 Yes, that's known right yes. Okay, okay. In this case, so I'm going to leave a byproduct of polymer we have a separate study on polymer which is also rather surprising and interesting but anything would look at this as surprising, but in this case we're 15:20:50 going to be talking about growth and looking at the moment, no growth and yeah and they both grow in them. Yeah, right. 15:21:32 right, the grocery do five was already a distributor five does not grow on group that already told you, and it grows on acetate. 15:21:39 Okay. 15:21:41 So, immediately we have this simple scenarios broken that continues to increase ascertain ammonium is taken up by through the old. 15:21:49 Okay and just just show the structure of a group now. It is basically this part is a glucose. And then there is the acid, the acetate group and as I mean group. 15:22:02 And, and it's characterizing other species, other species reveal where basically you have an assignment group that comes in cuts out the SEO group just releases it right and and, I mean, that takes out a mangrove Sharma but making it takes it by itself 15:22:18 for itself others does not take, and so was this picture you can see him if you look more quantitative and look at the yield. So if you put in 5 million more glucose to see, well we measure, this is amount of a one on one supposed to produce, and we know 15:22:31 how much acetate produce if you and if you look at the University of five on acetate, this is how much will produce and exactly it comes for the, the, the, the yield of a cult culture for everything affects. 15:22:44 Right. 15:22:44 And so this is a case of simple commands on Islam, of a former acetate and ammonia across. 15:22:53 Okay, any questions to this point. 15:22:58 can only Yeah, very simple. 15:23:04 A will not grow on acetate. 15:23:06 Okay, that's the other thing okay so ah yeah it. 15:23:11 There are various species that grow by agents will not grow on acid. 15:23:14 It's as simple as like example will say that the photon lab is just like it's amazing, no synthetic synthetic engineering cannot get such a simple system, okay but just, yeah. 15:23:29 acetate acetate ammonia I'm not gonna matter anymore it's just saying surplus. 15:23:37 Yeah, yeah. So, so here's question what was what was the rate limiting Acevedo ammonia and the answer was acetate. Forget about ammonia it's about carbon. 15:23:45 Okay. 15:23:47 And, yeah, so, so it's a very clean cut system. And so claim that the basic people wonder why was studying. I mean it's just a textbook. 15:24:00 That's a simple case. 15:24:02 But on the other hand, I will say, if you look at so this, the asset class really is the simplest example right but if you look at, think back in the literature, whereas a really well described. 15:24:12 Right. The okay so this this experiment with Julian Adams and we would really establish acid across but even with that. Okay, that's going to be assigned reading in two weeks. 15:24:24 Okay, you'll see it's actually much more complicated that the system. 15:24:27 Okay, so we actually showed the one of the reason we will start to look more into this was that we realized actually there's not a single very very simple okay so naturally occurring, some of this seems to be there and so we just bought one knows and 15:24:42 studies show the first thing, and perhaps the most important thing that we determine the capital, brother said was that well, we were doing these experiments in laboratory condition with a very high amount of a buffer. 15:24:59 Okay. But in the ocean. 15:25:05 Nobody's adding 40 million more of a buffer. Right. Instead, the buffer is a bicarbonate. That's a natural decentralization of carbon dioxide, it was, it was OK. 15:25:13 And it's about $2 million by carbon. 15:25:17 Two Minute model is now very much right so means if things are excluded two millimeter range, then you start buffer start to run out. 15:25:23 Okay then, other things. 15:25:27 So, a couple of study the system was a two minute buffer. 15:25:33 Right, so the expectation was that, okay so since the buffer level is low, then at some point, acetate become a certified some medium. 15:25:42 Right, and it becomes toxic. 15:25:45 So then immediately will have scenario in mind that, okay maybe it's not just conventional acetate is actually toxic and it will inhibit the growth of a one on one, and acetate is taken up by Studio five so have natural very natural classic example of 15:25:59 a central. 15:26:03 Okay. And the listener we had a line, I think it would be for every theorists immediately have this picture in mind is that, well you have one. So, as a functional Ph. 15:26:15 So you have a producer will be hit by a pH and then the consumer will then will will rescue the pH and the somewhere there'll be a stable at this point and then when they live happily ever. 15:26:28 Right. 15:26:28 Okay. 15:26:32 So, come to actually measure this clip. 15:26:34 All right. and, well, they don't cross, cross. 15:26:41 Okay, maybe this is a 50% chance to cross the channels that will now cross. 15:26:48 Okay. 15:26:50 So, while while is always going faster than reveal five. 15:26:55 Right. 15:26:56 Yep. 15:27:00 This is, this is a measurement in a static condition. Yeah. 15:27:06 Well, he's American, but not in chemo stat but then in as much as in batch culture mimicking us. So there's a range where you can arrange the parameters without changing much. 15:27:18 Okay, so why why is always going faster, right, and then so you can easily plug in a consumer research model we've measured all the years and everything already just plugging in and see what we expect and expectation is, what initially. 15:27:33 one grows and it will be excluding one more acetate. Initially, the three to five can take the acetate but since. Why no one's going faster there's no way it can it can take a border on the acetate right so eventually there's going to break up break open, 15:27:47 and that will be a point where everything crashes. 15:27:51 Okay. And if you show he mentioned a co culture and indeed that's what is. So, it's predicted that in six hours will crash and indeed in six hour, the cultural clashes, and that's where sort of a Ph. 15:28:06 drops down that will correspond to acetate going up and go Connect is not all consumed. 15:28:13 Great. So then, it's just a couple of system. 15:28:17 That's that that that that that's it, the end of description. Yes, there is maximum growth rate is when you give with plenty of glucose, of course in your dilution cycle cycle you always get changed. 15:28:31 Yes, yes, yes. So in this sense, there is no mystery here is that those two lines do not grow in the ocean maybe they can. Okay, okay. So, so, yeah so so that is in the ocean, whether whether the scenario, ever, ever come up, I don't know, it's a complex 15:28:53 situation for transmit they could produce, they could have a lot of carbon, but probably not for wrong. 15:29:00 Okay. But, but we're not all of this as individuals just studying the test. 15:29:06 Right. 15:29:08 So, yeah, we do not expect photography. In this case, I just simply simply one is always going faster than the other. Yeah. 15:29:25 Yeah. 15:29:28 In the standard in the standard ocean, I see what a. 15:29:40 correct. 15:29:43 Correct, but it happens both. 15:29:46 There's no reason why 15:29:50 it doesn't go down, it goes as as asset to increase pH lower right grocery have a goes down grocery bill goes down. 15:30:01 No so as you go this way so go into this low pH as pH reduced as pH is reduced. 15:30:09 All right. 15:30:13 Okay, so then, so. So Jeff score told us. So, so it's a very boring system, you know you you you you do the squirrels dilution cycle, you just you just see things wash out. 15:30:27 Right. And so, me remind you, this was the prostitution Sacco results with a strong buffer right and we can answer was on this established steady state. 15:30:37 So we'll repeated this experiment with this week buffer to minimum of. Right. 15:30:43 Right. So we do not expect anything. Well, of course, its finest way to close it so that's the that's the puzzle, will be address. 15:30:57 Okay, so, so, so this is a week by week, week before result will have two times be per. 15:31:06 So roughly still went to one another. 15:31:08 And so we sent our minister to wine for comment, and she made a point now you have mutations going on in there. 15:31:17 Right. And and we took warnings common at heart and we repeated this experiment with. 15:31:24 So we do multiple. 15:31:27 These gross dilution cycles and took the students at the end and repeat that exact same Terry Gross dilution cycles you mean effectively like a torpedo stamped, but diluting by a fixed no no rose is a very easy, as, as we understand how the others are 15:31:42 doing. 15:31:43 The you, you just, you take some fresh media, you put your culture into it. Okay, let it grow for some set amount of time, 24 hours 48 hours in our case of 24 hours. 15:31:56 Right. And then you take this siloed, you take the end result after 24 hours polluted. Let's, in our case for the fall into fresh medium, and just repeat again. 15:32:10 It's coming. It's coming. 15:32:12 So you don't care about the OD when you're doing the dilution it's a way of course we measure everything. Right, right. Yeah, I'll tell you what, but just by looking at growth solution. 15:32:31 it's like a chemo stamp but the dilution happens every 24 hours. Yes. Quick question this this is Alfred. I'm wondering what was a week buffer it's a $2 million. 15:32:37 The strong bathtub was, what more clarity. And I was wondering if you tried other buffer substances at the different is it the concentration or the type of buffer that matters. 15:32:52 I'm not hearing very well in this room. Is it the concentration or type of buffer that matters so if you go, you have a week buffer a $2 million. If you try it at three with Tris or some other buffer does it change the outcome, right, you know, where 15:33:11 I'm not sure how much capital has tried, we don't we don't think that type of a buffer matters, but the tap of a button matters in some case if you're not careful, if there is problems and know things like that. 15:33:12 did $2 million buffer. Yeah. 15:33:25 I'm guessing, if you avoid this problem, then you see this begin by carbon just the simplest avoid these problems. Yes. 15:33:32 The reason why I'm asking is that co2 might be also used for anabolic purposes, whereas, this is not 15:33:45 for Anna, yes. Okay, yes there is there is a co2 of course has more more more usage for for buffer but then we are not. I mean, even in other buffer we're not rushing out of co2. 15:34:02 Right. 15:34:02 Yeah. 15:34:04 This is aerobic system, don't see it isn't is needed but not very much, I don't understand, maybe you can just say it again why you don't expect coexistence if you're if you're resetting the pH every dilution cycle. 15:34:18 Right, which you have to do a large number of experiments to see divergence or something like that, right so so so then, then, then what we chat, was this is. 15:34:37 This was a six hour dilution cycle. Okay so because we know on the six hour we think we understand what's going on. Right. And so the same system with a six hour cycle Dan, as expected. 15:34:43 This this studio planet just disappear. 15:34:50 It's still the same dilution but just six hours we know six hours of what it takes to to to wipe it out. Okay so this experiment, then tells us that this has to do with what happened beyond exponential growth. 15:35:11 Okay, can you tell us. Do I really have a hard time hearing this, I'm just curious why so for the ones that you know the one that's less frequent than the 24 hours six hours just crashed. 15:35:22 The 24 Hour Workweek is but if you change the regime, at the third cycle, for example, to the six hour whether the crash again. 15:35:33 Tony exactly like it gets get slower and come back up right so while you can see this is a linear scale. Exactly. Yeah, we were able to understand all these features. 15:35:45 Okay, okay. 15:35:50 All right, but but this is just establish if there's only exponential growth in the game there's no, just doesn't really count as we. 15:36:01 All right. Right, so, so then that this, then put our focus on the, on the what happens beyond exponential growth, the stationary face. 15:36:12 Right, so some weird things happening some some some weird thing is natural to have us in traffic is that is happening in the stationary things beyond the growth phase. 15:36:23 Okay, so then we look at some more carefully look at the culture, in this case, we're looking at the viability. 15:36:30 Alright, so the first thing I saw was that actually after, after the end of exponential six I was the end of the exponential growth phase for parameter. 15:36:40 You see that one on one. 15:36:47 Just crashes greatly just it me the best way is like the best time is like a half an hour. It's just crazy okay just dying like crazy. 15:36:59 Studio five state but. So, oh and then here's some and this this test has to do with the medium of the pH so if we transfer. 15:37:08 So it does unfortunately it does not require culture so we look at the model coaches the monoculture effect. Right, so we grow, just a by itself, and a transfer to low pH, and the normal pH, and it's only when you transfer to low pH, he died at a similar 15:37:23 right. 15:37:24 So it's not like trivial like killing them. 15:37:29 Right. So, that immediately was oh, this is what's doing right so then it already, motivated, that. 15:37:38 So, there's one thing about license that can feed. 15:37:42 Feed the partner, the other is, even without license, this is a way just to delay things right and it'll get the time into right scale. 15:37:50 Okay, so then we develop another consumer resource model, including the test, and again we know all the parameters are countries, countries. 15:38:00 This is the situation. And yes of course you can reach us them stable state. And then, in the state. 15:38:07 The key feature of this state, is that it actually has a group net remaining okay and that's so how how it does it right so your basic decides, you have a, you have a freedom in the in the face and where to stop. 15:38:19 And when you're stopped as you have an extra stuff remaining. 15:38:21 Okay, and. 15:38:24 Okay so, so, so then, so then basically arrangers into a situation basically that with with with by stopping it at this point. Then there's just in the status that gives you the extra time for when I went to grow so so that it catches on fire. 15:38:39 Okay, so is this what's happening to now the answer is no. And the reason is that if you look at to the, the actual experimental situation, you see that all of the gluten so consumed. 15:38:55 Okay, there's nothing there's nothing left over. And actually, actually if you look at the, the, the two species that are about one to one to one, whatever. 15:39:06 It's close by this. It's not right it's not doing it by lowering the count of one to one. So, even though we saw while when death. In the first cycle, in a steady state in the stable cycle it avoid the one one way about it to die. 15:39:20 Okay, so you see that in the. So we start with some one to one ratio and you see why I was immediately after the first cycle and died a lot. That was the big That's right. 15:39:34 And after that it was able to recover and find yourself a state where it basically the two bolts of it and when I avoided that this. 15:39:46 Sorry, should I should understand this last slide is saying if you would look within a day like in your middle finger I would not see the death of a while so in essence a stable of cycling the model. 15:39:58 Yeah. And so you will, will grow until at some point basically pH lowest to a certain value, and then start dying right so that's the debt Right, right, but but in the experiment if it's that's not in the first cycle he does this, but no, not in a stable 15:40:14 cycle. Gotcha. Thanks. 15:40:19 Yeah. 15:40:19 The first cycle is density dependent right if you started was too low density, this one work, it will never take off, is that correct. What will never well oh no no no, he will was even if it started low density. 15:40:31 Oh no, it will rise even if it started below density. So then you will expect the cell death is so little that cannot take the culture to the, you know, to whatever to show your staff or whatever so there will be a point where there's, because as growing 15:40:43 faster than be, it just generating more acetate and be can take right so at some point pH is going to crash that that's basically where the game stuff. 15:40:57 Right. 15:40:59 So how does it do it. 15:41:02 So the best thing is, yes there's definite beginning right but at the studies in a stable cycle actually avoid to die. 15:41:12 Right. And as just to mention these the words that I think used in Joshua paper right so there's a community buffering effect by so by single species we see that the acetate crap pH crashes and all that, but somehow by growing together, they avoided get 15:41:33 rid of the acetate and the original the original double. Okay, so then we decided to look into the dynamics in the stable set. So again, it takes a few cycles to to get into basically a steady state steady stable cycle and. 15:41:45 And so then we basically then capital did the careful experiment after five cycles. 15:41:52 So, this was the first thing so so so so this is a very painful experiment, collected collected data from various time in a 24 hours, right. 15:42:02 The so so they measured the the the cell. The CFS the viability accounts. Right. and the first thing you see is that, well, or in a stable sample after the crash. 15:42:14 Okay so, so this is a, This point around 12 hours is where basic rules stop. 15:42:19 Right. And after that you see. Actually, the oil remains viable triple five will always remain viable but 15:42:31 he was stable cycle. 15:42:35 Then he samples for metabolize. 15:42:38 Okay. And amazingly shows right around the crash acetate we're going up so this was the pilot is that what we expect. Right, then within two hours, as it is. 15:42:55 And then the Brooklyn actors are just doing it is not it disappears as as as it was, was increasing. 15:43:06 Alright, so then this this Imperial acetate of course that explains the viability because there's no no no no acetate so it's the, the death is requires lots of acetate low pH, so just remind you, the transfer to sit model string transfer to normal pH 15:43:20 there's no that there's only a low pH, but if it is sales assaulting know PhD done. 15:43:30 Okay, so then, and then there's one more interesting feature the beginning. This was that was so far tell told you what happens after the crash after the glow start there, then actually before the before and after dilution is also interesting thing going 15:43:46 on. 15:43:47 You see that three to five is growing. 15:43:51 And what I do is actually not growing. 15:43:54 And that. Whatever the cause of this is certainly contribute to the overall stability because it hadn't give three to five more time to grow, it's just growing slower. 15:44:02 And you needed to grow the management to take up the asset. 15:44:09 Right. 15:44:11 So, then we zoomed into this critical period right after the crash. 15:44:18 And so you see, again, this room in beautiful times zero is where coconut drops to zero. Right. And you see that acetate was the heading this way up. And right after the crash and it's now going down. 15:44:34 But more than, more than half a day, then capital found that actually the other stuff that's coming up going. 15:44:43 So this other stuff. And we're talking about, you know, really substantial amounts of this other stuff. OK, now we're talking about major internal metabolites that should not be in the meeting with. 15:44:54 These are. 15:44:56 I wrote a and that's a good to me. The Goodies. 15:44:59 Can you remind us the concentration of acetate that's required to hurt the mouse remember them all so with a minimal buffer pH drops to five around three, a little bit over three minimal. 15:45:11 Ok. 15:45:15 Ok. So, whoa see like this other stuff getting tweeted. 15:45:22 This was a measurement, done by HPLC which we have in our lab we can monitor you know see doesn't metabolize. So then we caught up our good friend that we sour, who has gotta blow makes we can measure all these things are lots of metabolites. 15:45:40 And yes, lots of stuff as theory. Yes. One question, the fact that the red curve gets sort of close to the danger zone before turning around. Yes, that's it mean that this one over 40 factors that that human factor is just happens to be well match to 15:45:57 24 hour time scale. Yeah. 15:45:59 I mean, there's several cycles to adjust to getting into the situation where everything is just online. What does this happen. 15:46:07 But, you know, if we change. If we change the form factor then there will be other kind of arrangement, so this I'm describing me it's very painful. And so we mostly for this one, dilution factor but capitals also check a few other dilution factor. 15:46:20 The initial integration, I just I just want to make sure that the reason the record gets to about three before falling is not some biological regulation oh no no no no no I mean initially was not going through the roof. 15:46:40 Okay and then there was some adjustment, and then managed itself to just be at that point where the basic ask, is running out. It gets to a region where it starts to basically do bad things by. 15:46:44 But then, and then the other guys able to take profit. Yeah, 15:46:55 I guess in a very related sense supposed to cheat the cycle so after five cycles once it stabilizes during your dilution you add 10% more of strain a fresh trainee that's not adapted of restraint be that's not adapted does it immediately crash the system. 15:47:10 We haven't we haven't done that particular experiment, but we what we have done is no change the default, the initial the parameters we haven't done the experiment. 15:47:20 But yeah, I just. The reason I asked maybe as a follow up question is, are you basically selecting for particular metabolic strategies within the sub population so suppose training grows, and so long I'm not suggesting anything I'm just trying to get 15:47:31 my head's up, what's up what's down, okay, right and then later I'll give you my interpretation what's going on, but this in this cycle. Yeah, as against in the first cycle. 15:47:49 This presumably much more be around at the point when the looks like runs. Yes, yes then won't be around until it's doing this enough around, very quickly. 15:47:55 Yeah, so that's you, that's you know that has to do with the initial range would be to grow. All of these little things okay. 15:48:02 Yeah, okay. 15:48:05 And. 15:48:06 Okay, so, so okay so you have excretion of these pipelines and then you see the timing wise so now back to the growth curve again. 15:48:14 So this is assuming in the growth curve in in this critical region. And you see that be get to a certain point, and that there's maybe a small lack then basically during this window. 15:48:26 be steadily growing even faster than initially okay and that growth and B is consistent with what we know if the monoculture we feed be was acetate but also by ruling last night and all that stuff. 15:48:38 So the implication is that if the growth of the be right that basically took away. 15:48:48 This stuff, including acetate to raise your Ph. 15:48:51 Just Just wondering, we just the metabolites that were found were actually not consumed as into the two classes one one class that was consumed another class that was not consumed. 15:49:04 What are they chemically the non consumed. 15:49:07 We see I mean yeah so the other stuff that's a consumer not not consumed. 15:49:17 Make sure have you look closely at the rest maybe tell us what how we supposed to think about. 15:49:22 But, yeah. 15:49:28 Right. 15:49:30 Right. So, so then the suggestion on this point is that this other metabolize, a helping trivial fight to overcome this problem that otherwise would not have a look. 15:49:43 Okay, because in a sprint cycle is just out of shape. 15:49:47 So then the next thing we did was to directly touch this. 15:49:51 Right, so this is an inference made from a cold culture and all kinds of things happening so that we reproduce this situation in modern culture. 15:50:00 Alright so direct test of this class leading hypothesis and okay so reminder again the situation we're looking at right so then first we look at three to five that's growing in acetate at low pH so we would imagine it just looking at three to five in 15:50:17 in this region, it's soaked with acetate low pH. Right. And we'll see what can so Canada acetate within a without this extra help. 15:50:28 And this is the data. 15:50:31 Can I ask, what can you remind us just what their growth conditions were before you mix them together. 15:50:37 I'm curious about the physiological state of the cells when you're initiating the CO cultures. 15:50:43 Ku crossing the repeat. So I'm curious what the, the initial physiological conditions of the populations were when you mix them together. Can you remind us how they were grown leading into July we always grow them exponentially in the middle of exponential 15:51:01 phase, we start. 15:51:03 So, that was 15:51:07 your cycle is there as it is there acetate present in before you mix the cells together, but. 15:51:13 So this is a stable cycle so so then what what is happening is already at the end by so so so already at the end you can measure there's, there's a way but I'm wondering if you could get to the stable cycle without the initial crash, I conditioning, the 15:51:27 populations in the correct way. 15:51:29 But you're asking. I will read, read, read, read, production server know in a car culture. 15:51:40 Ask later. Okay. 15:51:41 I'm now hearing very well on this. 15:51:44 So, So then, so we look at review of our modern culture. 15:51:49 Okay. So, essentially, so so growing. Okay, the, at some point, the time zeros were transferred to low pH. 15:51:58 Right, very low pH, then you have to situation one is with this additional metabolite exactly this concentration on glutamate and the federal aid in the stuff. 15:52:08 The other is without. 15:52:10 This is with the, with the supplement and this is with them. 15:52:24 And of course, you see, if you measure the top I see that, well, for those that is growing then acetate will go down, it's mainly still growing acetate. 15:52:35 And, and the other metabolize going down as well and the pH. 15:52:39 in this one case with, with the addition of the sub metabolize supplement that your resource. 15:52:46 Okay so, so, so, so this this is a direct test right, or whether this additional metabolize has to help the benefit beat so this might be by itself. Yes. 15:53:00 Do you understand the mechanism of this extreme sensitivity of acetate acetate of your a story how how general this extreme sensitivity to acetate of your a string the one we start dying so rapidly. 15:53:13 When the acetate why the death, right, why did this way we know generally so so so the I have. 15:53:24 this is a follow up study of we finished studying the assets. 15:53:36 And then we're studying this, we know quite a bit of ice. Yeah, just just give us, we do not know why. 15:53:44 Oh yeah, we understand why why acid things doing this. Got it, okay, I'll give it a little bit of a test me. 15:53:50 Terry. Yes, just it with maybe is the answer just biggest question but I assume that the answer with acetate was the pH right you secrete acetate makes well Africa has an effect of pH but actually that took me in a half hour to explain the acetate part, 15:54:06 it's a non trivial problem okay then. Okay, I have no question. 15:54:12 Alright, so then the other part is what about one l one right so we what we saw here, is that these, the other intermediate metabolites helped three to five to grow, despite the low pH. 15:54:26 Okay, now. 15:54:29 Now how what about one on one which is supposed to be where we were thing in a central scenario that's create that's generating this stuff. 15:54:37 Right. And then so then capital this this experiment that normally you know he should really be doing this in the chemo stand again to mimic that situation. 15:54:46 Right. But then he laboriously implement his manual chemo step. 15:54:52 Okay so that is a show so you you you grow this in low, low pH and the as, as the pH is start changing because as it is excluded, right, then he would just kind of a piping, a buffer to kind of reflect anything pH in this link. 15:55:08 It's very effective. Okay. 15:55:11 And because we do not want to deal with chaos. Okay, wait for this kind of a weird thing so. 15:55:19 All right, so then what the define what we have you found that, looking at continues to be consumed acetate continue to be excluded, and this other stuff is also excluded. 15:55:33 Right, so that's kind of we'll try our best to replicate each part what is doing another story, so that these results and why on one under this condition. 15:55:42 Right, is it's not going. Okay, it's not growing, or the is constant, but it's still taking up growth neck, and it's converting growth neck into this stuff, which helps rebuild. 15:56:01 Do you understand why some of the some of the things that are secreted grow linearly with time and why some are non nonlinear. 15:56:12 Like, like, can you like can you predict, is it is a it is a me. Okay, so you guys interested and stay for another half an hour tell you about the acetate story. 15:56:35 Okay, I know you appreciate what's going on with with acetate okay but generally the the the transfers have a gluten is more difficult but for for for the band stuff is good to me, it's more complicated. 15:56:39 Yeah well for lactate and apparel V is this kind of basically just come with a group neck into this one. 15:56:50 Right. 15:56:51 Right. So then we come here than two major conclusion of this law is established experimentally. Right. 15:56:58 This is a really will say cooperatively that's induced by stress is only under When, when, stress is excluding the stuff and the stuff is helping trivial five to grow, we're we're in cannot otherwise grow right and the result of that growth is to take 15:57:13 away the acetate and, and ultimately want to one benefits by avoiding that. 15:57:29 Right. Alright, so, so a little bit into into mechanisms okay why, why, why is the acetate creating this problem. 15:57:37 So, so, let's say we first have a normal metabolism of So, looking like a broken neck is like glucose which is coming in from top of a consciousness. And it's doing your thing, and I see the gospel spread out at the very beginning. 15:57:55 Okay. So, I see you guys is a small neutral molecule, you can cross neighboring freely. 15:58:02 So whatever you have outside you have inside. 15:58:07 Right now you have a situation, if there's a pH difference between inside and outside. 15:58:12 They'll be a big difference between acetate or concentration. 15:58:16 Right, so just to putting some number. So do you can you can do this just so kind of working out the acid base system. And so basically the ratio of assets inside and outside is given by the pH difference between seven. 15:58:32 Okay, so, to appreciate a manager you see that if there's a, I have two units of the pH difference between inside and outside, then that means acetate differences 104 ratio and hundred four. 15:58:44 Okay, So if I have time minimum outside. 15:58:49 Right, I have a molar inside. 15:58:52 Okay, well what does the mole of stuff me inside right the entire entire content of a small molecule inside the cell isn't listening mode. 15:59:01 And this is probably you cannot beat. 15:59:06 It's a small molecule of this problem. 15:59:09 Okay so, and then this realization is not I work so so this this sort of thing was studying in the, in the 70s usually good stuff all studied. Understood. 15:59:22 Back then, right, and but then the current sort of community has largely forgotten about this right but then it's a very simple. 15:59:31 Yeah, Andrew. 15:59:39 Other systems where people have looked at this acetate access a week. 15:59:45 Internal buffers that will also decrease the internal pH and reduce proton motive force and can presumably have effects on ATP generation as well. So, we have looked at this and you need to give me another half an hour to talk about just that problem, 16:00:02 but at the moment. 16:00:03 You know it's there that's, that's for the hour why why why is causing this. 16:00:09 It's not really central to what we're going to say, okay, but yeah acid acid a problem is prevailing and very interesting. 16:00:18 Alright. So, the problem was. Now the Southfield was acetate actually there's also the asset is an eye on so the, the cat is potassium, or the field was potassium acid. 16:00:30 Okay, and all the stuff. Basically, it just kicks out all the useful metabolites. 16:00:38 And in particular, the acetate juice plus the student colonic. Right. And so our understanding is that since this metabolism the park, then click Next continue to be pumped down. 16:00:52 Then, click next continue to be pumped down. And, and it's just coming out as part of it and on that look like taters are severely and apparel they have one difference electron, but but then, otherwise. 16:01:02 So, it gets a little, it gets some energy in the process and it can keep this keep it simple. 16:01:11 Okay, so this is intrinsic effect of a, of a, of a weak acid. 16:01:17 Then, on the three to five side, we have similar things going on. 16:01:21 We have a. 16:01:23 We have a lot of internal acetate for the same mechanism, and that's going to basically expel lots of intermediate metabolites and stop the cell phone growing. 16:01:33 And in particular, so three to five is more so to to use acetate and used to run colossal reverse this group called Neo Genesis from acetate. Okay. And without these intermediate metabolites, it will be short of all these apocalyptic 16:01:49 right so now if you put these two together. 16:01:52 Right, so, then one has a scenario where reminder that Carlos gets plugged in here, and then parole day has excluded. And this is just perfect to compliment. 16:02:18 This guy was nice to me Can you explain the last slide again, I got lost, there was too much metabolism Can you go back one slide and say everything again. 16:02:20 Yes, starting with the neck. Yeah. Okay, good. Okay. That one. 16:02:26 I think I understood, you're saying it's short circuited Yes. 16:02:30 The Bounce, bounce there's like a basic and lots of declare that we measured all these things so as the larger the period. Okay, and coconut glucose are coming from the top. 16:02:40 And it, this part is of argument. 16:02:45 Is it a thermodynamic or why don't you know it's the big x the big x. 16:02:53 Most of these are reversible reactions. Now listen, we haven't tested every step but this is kind of a reasonable sort of a picture right into potential what's 16:03:17 just in separate space, you cannot, cannot get down 16:03:23 right now maybe you can do the next slide again. 16:03:29 All right. 16:03:30 So, same, same thing right you get approximate but normally was a normal metabolism is it takes acetate right and runs glucose Neo Genesis and through castration and go back up. 16:03:46 Okay, and again in the middle was was a high concentration of this, the guest is that all these know TCM a tablet intermediate muscle fibers fresh now, and it should not do this. 16:03:56 Okay, and I see, so you're basically saying that you're not producing enough energy to do metabolism so you secrete, all the amino acid glutamine Is that why it's not as simple as just by just by automatic balance, it just cannot hold that much. 16:04:13 Okay. Is that okay and then you there's no way you can get rid of this acetate, but it's just, it's a small molecule. 16:04:24 Guess what I'm confused about is why there's specific molecules that are secreted and others. The reason we were secret glutamate, even in our age PLC is because, which means the by far the highest concentration Okay, that's easy to understand that. 16:04:38 Okay, so then what will end up with a simple picture of a complementarity, where basically, you can use coconut oil was basically serve as the enzyme to to basically don't feed through to fight to do. 16:04:52 Sorry, just to make sure that I'm on the same page. So the picture is that the intracellular environment is crowded by acetate, and that crowding then forces out this sort of broad spectrum of other metabolites, maybe even roughly in proportion to their 16:05:03 availability intracellular Lee. 16:05:17 Is that correct. Yeah, yeah, yeah, yeah. Well, I mean it's not the cells are still growing so they're not completely, I mean they're still little bit we measure, Julie we measure that these internal Metallica the little bit of everything that and just 16:05:18 raised so much slower. 16:05:21 Is it clear that the numbers should work out such the Pirate Bay gets pushed into the B cell always or is that sort of a spin on or also show the number of workers basically can see how much, how much power comes out how much lactate comes out, the acetate 16:05:33 roughly works out, but was that always work regardless of how much clip neck is going in or is that sort of specific to have a steady stream of Arabic coming all you have to have goodnight coming. 16:05:47 Yeah, but the level is because you got one time, or the tournament spewed out. Right. And then, but but but to maintain sort of a qualitative study mountain for our to I mean, this is basically requires consumption picnic to see this stuff, but it's not 16:05:58 only the some of the smaller amounts of other stuff. 16:06:24 Right, right. So, so when we yeah that that's one other thing, a couple is taking up next, but it will yeah for three years, basically we just wanted to understand the system. 16:06:25 Yeah. 16:06:34 Right then, then a whole bunch of experiments that that suggested how general it is. 16:06:41 But, I guess. This makes a pretty strong prediction that it is sensitive to the amount of like knock you back down Luton So suppose you create a stochastic back dilution but you don't back diluting the same amount of black man, you start fluctuating. 16:06:55 and that means the system will also start fluctuating between various steady state. So it seems like you know that there's, there's a conspiracy conspiracy to line things up just was the right amount of looking at the remaining or, or of that right okay 16:07:14 getting to 16:07:22 shake it. Yeah. Can you just explain again why this cannot be done in a single cell in a single cell. Yeah, I mean, normally and whatnot. There's no way and they hold on to glutamate hold on to Pirate Bay, even say comes out there the artistry uptake 16:07:39 Why can't the single cell, why can't a solve the problem by requiring 16:07:46 acetate inside the cell. Yeah, well I think because because the single cell. Well, it can it can work under. Okay, so it is again. 16:07:59 You need to wait for my other habit it's all about acetate right yy acetate is doing, to some extent, so if you very acetate as a function, you know you measure growth of different point, grow Slower, slower. 16:08:11 Right, and and we can attribute the reason to the brochure down to number thing but you're trying to manage it tried to do it, try to do everything. 16:08:30 in and that 16:08:34 is related to Boris's question like, if it's running like Hollis is forward is like must be regulated not to be running glucose Neo Genesis, like if you can't really do both of those at the same Yeah, well, I guess, isn't it do, why doesn't it do both 16:08:50 of these processes in one cell. 16:08:56 Y kind of commercial model. 16:09:08 Ah, 16:09:08 yeah, Yeah, it says wait and what the code that's the quantitative question but by the time it feels that it's just way too high. 16:09:15 Okay, good. And the other question is is why does it do both things in one cell but that would be doing like colleges and glucose Neo Genesis. Yeah, none of that right that ended it will be it will be it will be I don't know how 16:09:29 it's a two way, it's a two way system it's a reversible we can only push one or the other. 16:09:41 Yeah, just, well, and you don't even have intermediate metabolites to do this because the other rates are low is are the reactions are fresh out. Well, 90% of the fresh now. 16:09:58 Alright, so let me go back to this last bit, which is the after dilution in into fashion media now there's no pH problem. 16:10:09 Right, so the okay so these guys this exciting thing for two hours problems are solved. Right, so while wants to be over just sitting there, no acetate no nothing right it's been sitting there for 12 hours. 16:10:20 No no stress. 16:10:21 Okay. Now, put into fashion media. 16:10:27 Okay and uh No, no stress on pH is normal, no had to minimum of a by car right and the gluten and everything. But while I was not going to do five stop growing. 16:10:40 That little drop in the. It's coming. 16:10:46 It's coming from there's a little bit of aggregation, but but the the, not much of it. But basically, one on one lab for about six hours just waiting for somebody to grow. 16:10:55 Right. And then, and start over. 16:10:59 So why is that because the the so that apparently says that if somehow he remembers, right, that because there's no problem at the, at the current moment. 16:11:11 Everything is normal looking at his day. But the previous experience seem to have done something. 16:11:18 Okay. And the other thing is if ya I was not growing well how does reveal five manage the rule. 16:11:24 Where does it get its acetate. 16:11:28 Okay, so here's this Thurman, again, it's kind of a reconstitution experiment. 16:11:34 So, you have growth of one to one. And so, this is natural, so it is a acidifying experiment, the environment pH drops. So then coupled took the culture of these two points where we're sort of where the pH is on its way. 16:11:54 Now, right, and then you just put these culture into fashion media. 16:11:59 But so, so, so in this case is just, just started the experiences so the problem. And this is one hour into that so the problem. 16:12:10 Took culture, these two culture into fresh media right Washington into the fresh media with the same thing as he would do in a culture. 16:12:19 And as a result, okay. 16:12:21 The one that's taking over here, just stuck, doesn't grow. 16:12:29 The one taken over from here, start going after little bit. 16:12:30 Okay, so that was our exposure. 16:12:34 Made it enable it kind of a kind of know about this and it stuck. 16:12:39 We had to wait much longer time before, before we recovered. 16:12:45 Okay, So what's going on here. 16:12:47 So there's a lack in a while and recovery, but just from the one to our exposure. 16:12:55 Okay, I'll address that in a minute but for now and see that even though it is now growing areas still taking up look neck, and it's spitting out, so. 16:13:06 Okay, so then that provides the acetate Let's reveal itself is stuck. 16:13:11 Okay, it's waiting for something in a minute what is waiting for. But then, in the meantime, we can still do analysis and can can explore the acid. 16:13:23 Alright, so, so then our thought was that okay so it's the exposure to a brief on a went to our exposure that's all needed by a low pH, that makes this happen. 16:13:34 So we're thinking that the Dow picture is that we know that during the shop or the internal metabolism of forced out much of the internal Metallica and so forth. 16:13:46 Okay, but then they are stolen by should be oh five. 16:13:49 Okay, so then after you put it into fresh medium. You gotta sell that doesn't have much internal metabolite it doesn't have much of a glutamate pool, it doesn't have a bunch of or whatever to grow. 16:14:04 Okay, so we tested this here, so we're focusing this part, focusing on a number of amino acid that we, we thought that might be the ones that are really it's depleted and measure, before and after the accident shocked to see that we see that aspartame 16:14:28 So then we repeated this. 16:14:31 Yeah so so looks like maybe it's insured of acid after after this expression. 16:14:35 wants to go to a drop aspartame particularly dropped a lot. 16:14:38 So then, so this was experiment was one to our exposure to low pH, and we added various substances to see what doesn't help it to recover. Okay. And you see that just by adding a little bit of aspartame, it can recover much faster. 16:14:53 If you add the whole suite of amino acids stuff of course the current immediately recover. 16:15:05 But in the meantime, Carlos can still run. So Terry Is it possible the cells are actually actively pumping out organic and ions to try and maintain automatic balance, like you refer to it as leaking but there's an alternative possibility that in an attempt 16:15:18 okay so so again I need to address you. 16:15:22 Yeah. 16:15:24 Yeah, we, it, it is. 16:15:29 It actually didn't even depend on acetate actual job is give you so we constituted this the same by filming the cell when some other useless metabolite you generate the same. 16:15:40 Now, it's really just filling the cell was was unusable. 16:15:48 Krishna is active or passive, the exclusion is a passive. 16:16:02 Okay so, so this seemed to be. So this little lab is basically the class it has the one on one had a spilling out in some without but it is a class. 16:16:20 It lasted. So then, you need to, it can adjust quickly recover, but other was that cost avoided the massive dying right, which is a, it's not putting it 16:16:28 right so um, what is it doing then during lag using those metabolites like what why is it lagging, is it building need to have all the amino acid before I can make an enzyme and everything right, so you need to slowly build that building out to me know. 16:16:48 Okay. 16:16:49 So to summarize the cell we have a, what we thought was the simplest case of crossbreeding, I mean well at normal pH and snow is very simple just comments on the sun, right, and the basically the first thing we went around was well with, with the central 16:17:08 free fat, right, the first thing that went around was that well there's no reason that acetate it just inhibit anyone awareness reveal five. I mean, we always write down all models that way, it gives them as a fixed point right but there's no reason why 16:17:21 they why they should do that. Okay, and about once. Once this happens, right, then both of these girls arrested. And when this was arrested, then this guy starts to spewing out these intermediate metabolites, and these intermediate metabolites and inhibit 16:17:37 the acetate inhibition and allows reveal five to grow, and when it's reveal five growth and of course now It took away acetate and it took away everything so then there's no more toxicity to one on one, that's kind of the. 16:17:51 That's kind of the story. 16:17:53 And right so, then a key thing about the stable cycle is that it's arranged itself shifted and phase and stuff just so that group not exhausted, exhausted, just right, as, as a problem. 16:18:06 That's not trivial, I mean the the has to do some adjustment to to get there. 16:18:12 Alright so right and then so ready to start in the first quarter except for the need to, to build up with the internal metabolites Okay, so Terry. Terry I have one question. 16:18:22 Yeah, so the the the mechanism that you've pointed out here requires that one as one stresses itself out by producing too much acetate. Yeah, so so if you were to lower the Mackinac initial conditions to a low enough level, if you would, I would assume 16:18:38 that the whole cross feeding thing would go away. Yes, right. Yes. So, is there any evidence, all you increase the buffer in that slice never a problem, right both should work is in the particle degradation system that was originally studying what is 16:19:04 this what is the availability of black and act transiently does it ever accumulate to you know multiple millimeters or is this it doesn't I don't know in fact I'm in the rock on customer takes to Australia look at the. 16:19:19 particular to do with with the intricate kind of a wiring of these 22222 species. They're really not international I was, I was saying, the, well I don't know what happened in the political situation but I doubt it's building up to a three or four or 16:19:24 five minimal stuff, certainly not in the ocean. Now in a experiment, it could build up, maybe two millimeter range, but let me know but, so we just by chance, happen to see something that's that that's working that I think would be working. 16:19:40 Okay. 16:19:47 So, I think I missed something, how is it that you're a strain has to dump out the internal metabolites but then the beast strain can take them up when there are both exposed to the same level of acetate stress, like, a chef Don't be honest metabolize 16:20:13 the string. Yeah, so how can how can the B string take up the internal metabolites when it also has a really high internal acetate concentration. Yeah, social, that was my picture that so acetate by itself, or it because it needs to do all of this stuff 16:20:19 to populate the other the other metabolic intermediate you cannot do it because it's stuck with Joe, but then communion with Pyro Bay coming in, then that complements what it cannot do. 16:20:31 So there's enough pay rebate going out of a strain that it is possibly going into the be strain or is there active. Well, it's not know where to take that part of it right so we still have a group that it's, it's a group that shut down its role, and it's 16:20:45 stuck at the bottom. And so slowly they make this stuff and it comes out. Okay, so, so it's not being actively taken up by the be stream. 16:21:01 It's not being actively taken up it's just all diffusion coming out. No, no. The be stream taking up the Pirate Bay. Yeah. What is that process is that active process, passive eyes, I presume it's active process I mean if the know whatever however you 16:21:23 normally take up. Grow cells with acetate and Peruvian and stuff right and show however it takes up. Yeah, I presume it's after process as normally. Right, so, then. 16:21:26 Okay, we have this a basically a picture and say well, these are the elements that are needed so that we made a simple consumer resource model. 16:21:38 Right now incorporating all of these features. Okay, so I will hesitate to do it but then we have a graduate student of an issue. Now Allah, Hoo very boldly just take out Let me see. 16:21:53 So now, fortunately, a lot of the parameters already measured right but they're certainly details of like a during this little bit of a hell of what happens we could not have measured the no steady state parameters. 16:22:03 Right. That's right so so for those parameters he gave us few sort of roundabout guesses. 16:22:20 Okay. So, you see, the first cycle. 16:22:24 Okay. But, so some parameters, highly constraints Mr Ron, just run about decimals, when we incorporated what we think is happening is everything that I described to you, and this is the result of the simulation. 16:22:24 There was the big crash of a one on one, right, and then there's the shift to so then with the 1444 dilution won't start from a really low. Right. And so there's adjustment of face and by the third cycle It's lovely. 16:22:40 So there's really nothing magical just the stuff we're putting, and you you call it up and it works right so talking about a week to get it to the work and. 16:22:50 And so this is the end of cycle behavior of the cell densities and concentrations. And then this is experimental behavior. 16:23:01 Just kind of things just naturally fall into places. And this is the, you know, the, the, well I guess this is supposed compared to that and this is supposed to be just kind of the you don't need anything more. 16:23:14 and and so there's some intricate dynamical thing but the face of it just naturally takes care, you don't need anything special. 16:23:23 Okay. 16:23:36 And. 16:23:36 Okay, so this is a title pictorial rise what what's happening but basically while one is the growing on the screen acetate this access, that's okay. Right. 16:23:53 Right. And then you have basically two phases growth phase, and the first two hour session we face the switching between the two. And that's all it takes to others. 16:23:54 generated. So at some point, then this allows enough Pyro Vader, able to turn this axis back with, that's all this going on so you have a limit cycle that produces. 16:24:20 And so right so lessons for theory. 16:24:22 Okay. So to summarize, I think the most important point I want to get across is in this case we look at at least coexistence is not a steady state phenomena, it's usually supposed to think about as a dynamical limits. 16:24:26 I don't know The Okay, do not forget about stationary face. 16:24:30 Right. And that the easiest way to get a fixed point is not necessarily the one that nature uses 16:24:39 will try to show our model is to block is the teacher can reproduce the face for late but then it's a bit broken we're trying to reduce it to something simpler so maybe can can can kind of a general life to to learn more about interactions and the lesson 16:24:55 for experiment is really, I mean, in this case you know the the lesson is that everything is happening in that two hour window. Okay, and I just randomly sample something at the end, you're not gonna you're gonna miss everything. 16:25:14 You're gonna miss everything. Right, so expensive class meeting, that's happening when the cells are not growing. So that was sort of my initial thesis, it sounds growing happily right doesn't have to show why should they throw away it's metabolite but 16:25:26 in this case when his cannot grow. 16:25:37 be generous cannot grow anyway right and if we can help, it's if it's a benefit to somebody else will help solve the problem. That's a pretty good competition. 16:25:41 Okay for the, for whatever reason, it was very it can it can 16:25:49 And question. 16:25:52 Yeah. 16:25:58 Just a curious right so if you evolve a string along right you know spiritually structure the environment. Do you think it will evolve grow through a strain in the sense that way this also happened to grow slower would avoid right this death by acetate, 16:26:13 and therefore be able to achieve longer term. So, a lot of things we don't understand about this industry in particular, why is a throwing away acetate and ammonia, where could use to to to build biomass. 16:26:27 And in particular, why is throwing away acetate. And so our colleagues. 16:26:35 Actually so evolved evolved to the oil one, and it's able, we got it able to take up acetate and we analyze it, without understanding Yeah, without understanding that it's hard to rationalize the rest. 16:26:49 so curious. So when you say stress. 16:26:52 So it's not necessarily you know activation of stress mechanism it's, it's this imposing a flow of acetate that has to go somewhere, is that correct interact yes then we have stress, it's just that nothing is provided with everything right, it just cannot 16:27:11 grow. 16:27:12 I guess what one analogy I have in mind is what happened with, I don't know, cyanobacteria where carbon is not limiting comes in an overflow maybe an outcome of the fact that there is just, you know, endless energy and carbon, and it has to go somewhere. 16:27:28 So, isn't this the same here we have this acetate that is filling the cell is has to go somewhere, but they're saying it 16:27:39 doesn't have to go anywhere. 16:27:48 He, whenever he was I said acid and that said no matter how fast you try to consume as it will just more attitude, which is more see the actual just coming in the film. 16:27:53 But the so cannot harbor that much acetate right so you find us a way to adjust to get into our smart a balance me into this also internal pH problem that Andrew referred to right and so the number probably needs to deal with. 16:28:07 But that's another talk. 16:28:11 We're gonna say I'm curious if you, you know if you try to impose a constant flux you can post a flux of acid inside the cell will use okay so okay okay so so so so now if I was to switch to the other talk market tell you this, there's not much of acid 16:28:31 reflux. Okay, it's just the Dead Pool. 16:28:48 I know I'm almost done with some people I mean it doesn't matter i mean you know the. Okay, let me just finish them I don't know what else I have in my talk. 16:28:56 So, so the. 16:28:58 Yeah. So, let's see. 16:29:01 Okay, so I guess I should mention this a stress rating hypothesis, or it was a hypothesis raised by colleges. 16:29:07 20 years ago but when we're under stress, rather than maybe corporations turn out the self help each other, and the various kinds of experiments that people attribute to have this type of mechanism. 16:29:21 I think we've got one, I'm just really, this is nothing but but but that right the. Now, whether it's intended to do this, or whether it's just a result of acetate a stress, I don't know, but the fact is that it is. 16:29:32 It's only under stress that it's doing this so cooperation. 16:29:37 And another clear requirement i think is a really a complementarity of the modes of metabolism, so. 16:29:44 So the. 16:29:49 We know the cells are primarily running central metabolism downward, right from glucose down to the acetate and other ones the primary one. What's this, because it's a two way traffic you have to pick one or the other, right, and for whatever reason is 16:30:12 different organism. That's it that speculating a favorite book on your general favorite so when you have them two together. there is a possibility of a couple months. 16:30:14 Right. And so we think that this is a boy extensively happening then just under our autos pizza system. So basically it involves acid the stress and the exclusion of acetate is very generic, and I just pull out Josh paper in the supplements you see that 16:30:36 actually for for their community running. 16:30:40 This is a single species community as well. 16:30:46 In the in the situation, you'll see that there was no pH is in this range where all of this thing we're talking about should be happening. 16:30:55 And so, so my knowledge among and just Capella ominous as a phenomenal postdocs really did everything and just more and more stuff keep it will never finish this apply to just never finished, right, keep on going until everything is fresh now. 16:31:12 So very fortunate to have a couple, and then least lab, Sammy punctuality to the mass spec, and our hopes that it will not have started without learning about this ecological problems from us. 16:31:26 All right, thank you. 16:31:36 First of all, congrats to top heel. I happen to know him and he always does great work. 16:31:44 And 16:31:44 I hope you can hear it I don't know it's not clear he's there. 16:31:47 So, so just to, as I understand it, the point is that if you have weak acid that has a PK that's beneath the, the typical pH of the cell that you can have this kind of pump that you've described where the where the concentration inside is higher than 16:32:01 the concentration outside by a big margin. 16:32:05 So, and then but you also described that acetate is important because there's particular metabolic role right that it when when it comes in it sort of gets screws up the metabolism in a particular way. 16:32:16 So I'm curious if you've tried to decouple that in any fashion right like if you try lactate versus some other fit right like you can try lactate part of it other week acids that participate in metabolism and various other week acids that don't. 16:32:28 And I guess a related question maybe it's two parts, I don't know, it seems to me like it should also be the case that anything that has a PK in the right range, that's made intracellular you should just passively leak out of the cell in the same way 16:32:39 that you've described, right, that if you make a weak acid in the cell it becomes prorated it leaves, and that maybe this is just the basis of the extensive cross fading that Josh talks about in his paper. 16:32:51 So, yeah, a lot has a lot of what was your son had to do is acetate itself so we should just have a separate times, those are interested just talking about something. 16:32:59 Right. 16:33:01 Yeah, but then about all these other sort of scenarios that you mentioned some of us in animation. Yeah, so we are certainly on all of these puzzles right until they finally came together was probably just a few months ago, we didn't know what we're doing, 16:33:20 but once it came together the, a lot of a lot of thing was a capella was looking for a job this year, by the way. He's got a he's, he will be looking for army of people to run out to check out all this scenario. 16:33:38 I mean, I'm next. 16:33:41 No, no. 16:33:47 Your. 16:33:47 First of all, really, really neat story and I congratulate you, too. 16:33:50 I'm also wondering how you can ever be this effect. In other words, there are some microbes which can survive pretty low pH and it sounds like from what you described from the basic thermodynamics. 16:34:03 If you add some of the assets in the media in the in the environment and you have a pH gradient of, you know, two or more between intracellular extracellular then you are dude you are very interesting question and I think, in our study of this asset a 16:34:21 problem that is the most interesting question, sort of that that much right what, what does it take to to be the asked a problem. And so, an experimental side we are evolving, with very obvious is able to, to improve a bit but not very much it's not easy 16:34:39 to do. Right. And, but then. So, again, I would say, you know this. Let's find a time to discuss together and I'll give you my thoughts on this. 16:34:49 It takes time. 16:34:52 Okay. 16:34:53 So just some general comments I suppose the obvious ones in some sense but so I've seen that one of the main messages, is that even when things are simple they've really complicated, that is by far the sort of the way we send our management too many people 16:35:07 to get feedback, and the by far the you know the first response was, boy, it's really complicated. Yeah. 16:35:17 I think you know this experiment in Kevin shorts lab one evolving Eastern low glucose and boring conditions everything and then trying to disentangle what goes on through the growth pollution cycle, and these huge effects of single mutations that come 16:35:29 in. Yeah, and it's, you know, in the end it's roughly that they they recover from hangover faster than mutants and so get going faster is wanting it, but the whole discussion in terms of numbers of niches and things and counting resources and so on Amazon 16:35:44 law says how wrong, that all right as long as you've got these complicated things. This is blooming and depending on the exact. 16:35:54 The on the exact on the exact conditions, but it does enable many many more different things that can coexist potentially right i mean if you just have a. 16:36:02 If your phenotype your single resource and your phenotype is how you grow as a function of the concentration and how you how you how much you eat is a function of the concentration, and then you can in principle have as many types coexisting as you want, 16:36:16 with just the growth dilution cycle right so somehow the whole discussion of the the resource models and so on. 16:36:23 I just seem to somehow miss the all of the complexities are there, even the simple things, and we need to new, better ways of starting we want to take 16:36:47 a look at it. So, I wouldn't get upset if you hadn't. 16:36:50 So, I'm just going to take a different angle on the same question. Okay, going back to the beginning of the talk, you said you take strain, a, and then you take strain be. 16:37:01 And you didn't tell us really how you chose them and you you found this interesting effect. So should I therefore expect that any pair that I pick at random, will be interesting as that, or whether there are some 20, other pairs that you looked at before 16:37:22 this is a very little far when aim to picking the string, and Stefan definitely not like a planet thing we try to mimic exactly the natural condition and and all of that but but we did have, we did have like, you know the basic thing going on. 16:37:32 Okay, all right, yeah this is a small sample of it. 16:37:36 Was this the first pair that you pick. Yes. Okay. 16:37:47 I guess just to follow up on horses question to we we've been saying strains are these the same two strains of the same species if that Oh no. 16:37:59 Thanks. 16:38:03 Yeah, so definitely the first reaction is this is way more complicated than I would like it to be. 16:38:10 But can we go back to that original plot, you know, just the two growth curves crossing. This is like the easiest way to get stable coexistence. Okay. 16:38:19 Rescue something very much like that from the system by just incorporating the internal state. 16:38:24 So there's some effective growth that happens the the internal state is becoming bad for growth, it has to give up a number of generations. So there's some like effective growth that was done when I wasn't I wasn't paying attention to this is this is 16:38:38 a this is a curve you want right yeah exactly what about it, so there's there's some sense in which stream, a is doing a bunch of growing. Yeah, but then it's also its internal state it's causing it to lose potential growth in the next cycle. 16:38:53 So there are some like effective growth rate of us over the course of the whole cycle. Yes. Can it get me back a picture like that there's some effective growth rate. 16:39:02 Yeah, well, the, the model including path is exactly that right you you'll kind of, you can compensate effective grocery store, if you will. 16:39:13 Okay. But, uh, yeah but, but, I mean, how many people consider this situation, at least in my mind will automatically will just assume oh that's got it's got to be that when you see a coexistence. 16:39:28 Right. 16:39:31 But 16:39:34 I'll respond now. 16:39:37 But unless someone else did you go. Okay. 16:39:40 No, but I mean I think the question is again model what is it useful for what timescales Are you asking because like we also see dioxide everywhere. And we know we can model that. 16:39:49 So the question is just like you have to know the applicability of your model so if I want to ask over many cycles I don't see why I can't time average and things out right like the rigidity of this thing is very hard but I know there's a stress tensor, 16:40:02 so that's all we're saying consumer resource models are, I think you just have to know their limitations, they're not good for mechanism. 16:40:10 that's I guess exactly what I was saying, why you're building a model for right i mean whatever model we do these experiments so I consider it a success you were like why are they cross feeding and 16:40:24 really quite a package, but was also we were talking about class PT, right, he explained to me, his picture. And, you know, in my mind, I say I don't believe it. 16:40:33 So then we'll go into that but that's the only purpose of theory and some sense to tell you how to plot the axes and inspire good experiment I don't know, maybe maybe you have much more belief in theory, than I do. 16:40:56 In sound status right like what Daniel was saying the beginning right if you include the possibility of sound license and everything that that diversity. problem is, is like a noun problem. 16:41:01 In principle, cell has thousands of metabolites and it spills out in some way. And is it so that there's there's enough, as long as there's cannibalism going on but, but that so that we're asking sort of a know is that happening. 16:41:14 The what we show here is that the cell does not need to go so destitute right also feels like a system where no time average version of this explains stability. 16:41:23 It's only because of the cycling that you have step.