15:01:52 Okay. 15:01:53 Um, thanks. Thanks for giving a good introduction to the nice vacation. 15:02:07 Friday say that I want to say thank you. Your organizers of the conference, and a real are from linear perturbation taking me here on an airplane and getting in front of people for the first was really good. 15:02:12 So today I'm going to give a short talk about work that is in progress. About pathways putting in, you know, flying back to your communities. 15:02:21 And so this is work that I've been working in the past year so. 15:02:28 um so i think i think this question, or this notion sort of on people's minds, quite a bit, but I'll just sort of be explicit about what I'm thinking about here. 15:02:36 So, what am I talking about with pathway splitting Well, you know, throughout the microbial world, there are instances of metabolic pathways which consists of sequential reactions, whereby in various steps in those reactions you make various amounts of 15:02:54 ATP. And oftentimes those pathways can be contained encoded in the genomes of mutual organisms, and thereby the entire process can occur within one org. 15:03:06 And often. In other cases, the halfway can be split among different genomes, different org. 15:03:20 One thing we've been discussing alive came up in the last time it came up in Terry's talks, is that, you know, there can be excretion Francine excretion of metabolites which from which he can be derived. 15:03:26 So in the case of a full pathway there can be some amount of excretion of intermediate which can then be used by other organisms in the environment or then, or if, if, if the Spain is dominant or the fastest, or whatever, you'd be reduced by that strain 15:03:40 later on. 15:03:42 on. But in the case of split pathways cross feeding is really obligatory so you're kind of hard coding in the phenomenon that this this intermediate has to be excluded and then utilize. 15:03:55 Okay, so, I think we've talked about some examples of prosecuting. 15:04:01 So I think what is the traditional view of education, which is the. In total, it is the process of the conversion of ammonia, the reduced nitrogen compound to nitrate which is an oxidizing compound in aerobic conditions, and the traditional view of magnification 15:04:27 is that it's done by two organisms or two gills of organisms, and that the first step in this process whereby some, some ATP can be harvested is done by ammonia oxidizing bacteria so they do the process of converting ammonia, to a nitrate and or two. 15:04:46 And then this nitrate is excluded in the environment, and by right oxidizes do the rest of the stack. 15:04:54 And there has been some work. 15:04:58 For example, work of, so there's an interesting review paper by coaster at all from 2006 which kind of speculate as to why we see this as the dominant sort of natural fire paradigm in nature, up to the point of up to this point. 15:05:13 Complete natural fire we're in all the steps of the process for contain one organism and encoded by one genome. That was not known. 15:05:20 And so, so there has been some speculation about why. And, you know, these folks argued that, you know, how saying all of these steps within one's within one genome allows for one to harvest more yield per unit substrate, by, by, you know, doing all these 15:05:43 steps than one. So in, in an environment where I that is under section for yield one might find a complete Metro fire and indeed. Some folks in 2015. I think simultaneously found a complete match fires in these conditions. 15:05:51 I think simultaneously found complete match fires in these conditions. And so, so there's sort of a, this is my way of saying that there is a sort of a framework in which people are thinking about halfway splitting. 15:06:04 And it has something to do with. I mean some situations selection for the rate of ATP generation may be occurring. So perhaps you could do more, this step faster and thereby generate more ATP per unit time by splitting pathways, and in other situations 15:06:19 where selection maybe for. 15:06:34 Okay so sort of contrast the example of nature fires now bringing to bring the natural virus into the picture so separate It's been a long time talking about this. 15:06:44 So, we see split pathways Indonesian fires, as well. And the example that I showed in in his slides was of the sort of course grouping of strains into nurse pains which which, at least do the first two steps in the process and optionally most often this 15:07:05 this step. 15:07:09 Yeah, for natural fires have the competed at those like the three strengths, like the the whatever what's the one that can do both. And then the two by the two separate ones. 15:07:22 Compete them. That's a good question. I am not aware of if anybody is a competition. 15:07:28 Yes Okay so, Stephen described the example of nurse fans which do at least the first steps in the process. And also, in our screens which she the first step and then nurse James which to the second pass the additional steps. 15:07:42 And so, so we see examples of this we isolate strange from spurs and we see examples of these different phenotypes. Another study was set but also cited by like us at all demonstrated that there is a large number of these sort of modular genotypes were 15:07:59 straight different strains do different steps in this process. So, I think I want to make a circumstantial argument here, which is that these are all signs that were isolated from soils. 15:08:12 There isn't a good case to be made that in some soils, there was a strong selection pressure for yield and others set of servers have a strong selection pressure rate, but I think it's not exactly clear, for obvious that halfway splitting into notification 15:08:28 fits into that paradigm. 15:08:31 Okay, so what am I going to talk about today I'm going to tell you about three parts of this project. And the first part of this project is about trying to understand what environmental factors drive pathway splitting the education so if it's not some 15:08:45 way. 15:08:47 Clear selection for rate or yield what what aspects of the environment drives pathways. 15:08:52 I mean that's can be analysis of data. 15:08:56 The next part I'm going to talk about is sort of a laboratory attempt to think, to dislike for pathway splitting based on what we learned from step one. 15:09:05 And the third thing that I'm talking about which is very much a work in progress is proposed theory to connect the environment to physiology and selection. 15:09:17 Okay, so the first question that we asked was, what environmental factors might drive pathways putting for deification. 15:09:29 And to do this, we turned to what is really a nice dataset. 15:09:32 So, These folks 15:09:37 is folks in 2018 publish the status that were they, nice job of sort of systematically or broadly sampling the world the top sales of world. 15:09:48 And in addition to in shotgun marriage nomics, they did standardized soil characterization, so we learned something about the environmental characteristics of the soil samples. 15:09:58 And so for instance they measure. 15:10:01 Organic Carbon concentration, Ph. 15:10:05 And so this is a really nice data set for relating environmental factors to signatures in the one limitation that I want to emphasize about this data set is that it's a bit too shallow for Mac so this for the assembly of managing the genomes, better genomes. 15:10:26 So, you know, the question here that we ultimately would like to get to is, whatever viral mental factors drive the splitting of jeans within genomes. 15:10:35 And so in order to really resolve that using it Chaka metagenomics data that you need needs to be deep enough to assemble whole genomes and this data set is not really going to let us do that. 15:10:43 So I'm going to sort of dance around that limitation and try a different type of analysis. And I just want that to be there. 15:10:54 Okay so, so the first thing that we did with the status that was focused on the interpretation were databases. So these are the the you know the the genes that are responsible for the various types of magnification. 15:11:08 And so they're indicated by these different colors and different nitrate reductive, then I tried interest oxide. 15:11:18 And so essentially what I'm doing here is I'm looking and operating upon the relative abundance is just the demystification gene content. So I'm not looking at relative abundance within the set of all genes in the community, I'm just looking at the relative 15:11:31 abundance of these. 15:11:33 Each of these. 15:11:34 Some. 15:11:38 Okay. 15:11:40 So, when okay so what we did with this data set is first try to categorize whether there's any sort of low dimensional co variation here, in terms of the present, or the abundance. 15:11:53 And we did with something along the lines of PCA. And for the sake of interpreter ability we use negative matrix factorization. So essentially what we tried to do was resolved each of the samples into the summer of two different modes. 15:12:07 So, that's what these h1 and h2 are so these, you can think about these as like characteristic genomes, or characteristic unit types within a community, and each sample is characterizing as having some amount of each of those characteristics phenotypes 15:12:17 that's the weights W's, and negative matrix hatch ratio, they should not just means that these have to be positive. So that's the way in which this is distinct from PC. 15:12:27 And when we do this analysis. To cut a long story short, what we find is that there are two characteristic modes that we identify in this data, and the first characteristic mode or, you know, characteristic genome, you will, is a mode which which mainly 15:12:47 has a contribution from this nitrate reductive. And this is a particular nature that history, remember we had this discussion that there to share a doctor says for the first step and RG and that a and so this one is just the first RG. 15:13:01 And in the experiments and you know in our scan of the microbiology literature, you find that this is the sort of faster and higher yield nitrate reductive. 15:13:13 And so, you know this this mode kind of resembles the, the, the phenotype of a producer. 15:13:21 And then there's the second mode that comprises other electrification reductions. So now is the, the other nitrate per case. So, the slower one. 15:13:35 Okay, which is by far the dominant one in the status head, there's not a lot of interest and and nasty. 15:13:42 So this is sort of like a northerner strain this mode. It's rough phenotype and if it resembles I would say a nitrate specialist because it has the slow nitrate reduction, and I try every case, I say, slow nitrate project is sort of the capability of 15:14:02 producing my trip nitrate fast. Yeah. Is there any way of accounting for copy numb would like the typical copy numbers of these genes and genomes that somehow be biasing you make sir, some these conclusions for instance technology is usually found and 15:14:16 two or three copies as opposed to another genome defined typically in one copy right so you're saying, Yeah, but what one frequently observes, if you look at it completely and I'm database annotated see multiple copies and RG, for example, sometimes I 15:14:34 that is going to be. So it's not, there's there's sort of no characteristic rule that one sees always two copies of energy in the genome or something like that. So I think I think that would be. 15:14:46 So I think I think that would be. Okay, so we identified these two modes and I'm going to sort of shorthand call one of them in our mo and one of them. 15:14:53 For the natural fire motor and our nerve, 15:14:57 an exit we did. Now bring the environmental variables back into the analysis is we asked what what environmental variables are well predicted by these modes. 15:15:09 So essentially we can reduce this this data into these two ways. 15:15:15 So there's two coordinates for every observation and set up this regression problem, where we try and predict an environmental parameter. So in this example Ph. 15:15:26 from these weights. 15:15:30 The amount of these both sample. 15:15:32 And what we find. So we just do this by lls. And what we find is that by far. 15:15:38 pH is the best predicted by these modes, and the rest of the environmental factors are really not well predicted. Right. 15:15:47 And so what I'm showing here. I don't think I've indicated here, I 15:16:11 Okay so, um, what I'm showing here now is ordering is his chart by recent page so what you see very clearly, is that that naar teaching content is dominant increasingly dominant as pH decreases. 15:16:18 where there's more of this dark mode as each decreases. 15:16:37 Okay so pH is on the rise, as pH decrease. Yeah, this is, 15:16:44 This is, 15:16:52 I guess, can you can you say anything about the direction in which this predictor is actually working because it could be that in soils with low pH nitrate concentration is particularly high, and therefore Naji is a favored outcome, versus the having 15:17:07 high energy is what leads to low pH soils right like can you say yeah I think about that. Yeah, that's a very good question. So I think. 15:17:18 So you're saying is, so pH could be leading to a high nitric concentration. 15:17:25 So I think one thing I'll say, sort of backing up from that statement is that I think that there is some mechanistic relationship between pH and physiology the justification pathway, and I will get into that and perhaps your question will be answered. 15:17:40 And I'll get into right now so. 15:17:43 So, you know, the potential mechanism here is that pH affects the toxicity of nitrate which is the second intermediate diversification. 15:17:50 So, you know, as pH decreases the toxicity of nitrate increases, and there is, I must say, not a lot of physiological work on this phenomenon, but what is, what is believed to be going on here is that there's some on Chinese pronunciation of nitrate to 15:18:08 nitric acid, and also spontaneous conversion night, I tried to nitric oxide. And that can affect that metal centers of enzymes. 15:18:18 And I just want to bring up the physiology of the sort of spatial arrangement of the genes that are the enzymes and the unification pathway. 15:18:30 So, as Terry described. 15:18:36 There is a differential between the cytoplasm, and the external pH environment. And I think what has been observed is that a very positive is also relatively and buffered so it is. 15:18:52 So, one should view the positive is having the page roughly that is the same as yesterday environment, versus cytoplasm is, it can be relatively buffered up to. 15:19:02 And so what you see in the dash vacation pathway is that the first step, especially, specifically, using nitrate reductive GM that it happens in the cytoplasm so that can be referred to the toxicity of nitrate pH, whereas the rest of these enzymes are 15:19:27 not so protected. So, this is just to say okay we see RG being favored at low pH, and perhaps that has something to do with the fact that it's localized cytoplasm and perhaps not so. 15:19:45 So on the. 15:19:45 This is not susceptible to 15:19:55 I'll be just knocked up the PK it's not, it's kind of an in a similar range of acetate up so then if the South maintains a buffer the internal pH then you will you will have an explosion of nitride inside of so external pH drops. 15:20:18 Well, I think, I think, I think my argument is simply that if the cytoplasm pH is near neutral. Yeah, then you're not gonna have so much spontaneous pronunciation of I tried I know but but but if if the neutral species can leak across and outside is a 15:20:31 right. Uh huh. Then, then you just have you will just have a basically leaky pathway for night, and I tried to get into so I see, I see, and for the step night tree analogy, yeah i think i think it's interesting point I'd like to talk about. 15:20:51 Yeah, so, so. 15:20:58 Okay, so we're not before you go on doesn't wouldn't appear to this will also affect like the ability of the soil to retain these different nitrogen species. 15:21:10 So, there is some 15:21:14 chemical production of I tried that occurs at lower pH, but I think it's, it's not. I think the rate is far slower than what biologist. 15:21:27 Okay. But doesn't it also like run off like this is a big problem in agricultural soils, and I don't recall from the paper how many of these were excellent versus natural soils. 15:21:46 Yeah. Yes, surely handsome awesome though you said the possibility that you're raising is that there is transcending species of nitride, and then that could run off. And there is no exposure toxicity or the ability to utilize as soon as possible. Okay. 15:21:58 Okay. And so, I also want to say that. 15:22:02 So we're not the sort of first people to arrive at this epicene I'm thinking about unification. 15:22:07 I trade toxicity as a function of Ph. 15:22:10 So it's been speculated that splitting up the metabolic pathways into different cells can alleviate the transit and accumulation of nitrate. So, this is similar to the rate argument. 15:22:24 So Eric can, if, if, if a cell is constrained and the resources back in I can devote to doing each of these steps in the process. 15:22:35 The individual for the National Fire made transit accumulate nitrate and pathway or to an hour plus an hour stream video, reduce the accumulation of the intermediate, and thereby led by just want to bring that out there. 15:22:55 I just want to bring that out there. So, that's not the hypothesis that we're going to pursue but I just want to. Can I can I just ask really quick. 15:23:02 Separate kind of glossed over this part in the previous talk but there are some transporters involved right so that uptake these various things. But a lot of the biochemistry happens in the pipeline ism, and I've never understood like how I'm supposed 15:23:13 And I've never understood like how I'm supposed to think about the permeability of the outer membrane. 15:23:16 Right. Is it very different from or very similar to the permeability of the inner membrane. 15:23:21 In other words, like, you know should I be thinking of these organisms is like mostly holding onto the nitrate unless they purposely expel it or is everything exchanging with the extracellular space at similar timescales like. 15:23:32 Does it matter what the charges are. 15:23:34 Yeah, that's a good question. I think there's not a lot of work on it. 15:23:42 Okay, so we have some idea that page is somehow responsible to halfway spending the night. 15:23:59 And so what we did to get at this was to take six soil samples, which were gathered by members of our lab to extract sort of communities from these samples and to securely propagate them in demystifying conditions at two different pH conditions on page 15:24:19 six and teach them. 15:24:21 And we do this with modest dilution factor of 3d cycles for 12 cycles and sort of the conjecture that we started with was okay in one case you see at low pH Do you see our listeners trains, being the fittest and leading the I tried toxicity and an entire 15:24:53 to you know somehow managed to keep the pH. 15:24:55 you see potential buyers interest 15:24:56 They are the initial pH and be added pH measurements at the end of the first few cycles and the pH hasn't changed very much so there's a 40 million more phosphate buffer which and international and conditions, the strains are not producing so much biomass 15:25:24 So, yeah, sorry, your question. 15:25:28 God is low. And so, and so the app right and the end the God is 1.1. 15:25:38 Yeah. 15:25:38 Okay. 15:25:41 So, at the end of the experiment, what do we see so we expected to see maybe split pathways but that's not what occurs. So, what we observe is that there are, and this is based on Shaka metagenomics and assembly of genomes genomics data, we observe is 15:26:01 full pathways. Overall, and we see sort of characteristics brains are in each different pH condition. So, what we observe is that there are different strains like two different pH conditions but they're both both natural fibers. 15:26:19 So I think the failure here is interesting. So, what we observed. 15:26:25 And this is based on predictions from our, so we assemble these meta genomes, or this you know, managing them, then we made predictions using these statistical approach to describe 15:26:38 what we observe is that, primarily, we select for, I tried specialist at high pH. 15:26:56 And that's computed based on the rates. 15:27:02 And so what we observe is that I tried specialist here to dominate at the higher pH. 15:27:14 who 15:27:14 would this segment about the enrichment selecting for full pathways, is that equivalent of saying that if you were competing a nurse strain and then our strain in in a nitrate environment, then you select more often for the nurse train and the nostril. 15:27:19 I trade specialist 15:27:36 Yeah, so the thing that I didn't quite expect is that our assumption in isolating or not isolating but extracting these complex communities that there's going to be many different phenotypes including our and our phenotypes, and that the end result of 15:27:48 this initial experiment which started with complex stupid types that you get folding chairs and bookcase 15:28:02 things on the next slide. How am I supposed to be seeing that there are full pathways is it that like one street one families kind of dominating or yeah so there I won't say directly from the side but, so I'll show you. 15:28:20 Essentially, get. 15:28:25 So, these are the characteristic phenotypes of those strengths are, but so the blue screens in that previous button. 15:28:35 The symbol the mags for those trains, when we get to types that look like this. And then we assemble the max with the red screens, look like this, that's where that comes, I see there's at least one species that has the full pathway. 15:28:45 That's right, yeah. Okay. 15:28:52 So I want him. 15:28:55 bridge to propose theory for what might be happening here. And to get it that I first want to use the approximation for fitness. And so, Stefan described this consumer resource model which dramatized metabolite dynamics in her the extra fires. 15:29:12 In, or the natural fires. In the previous work. And so we're going to use that framework and simply make an approximation. So, in the limit where nitrate and I tried are far greater in far greater concentration, and he's going to do primary, which which 15:29:29 step looted to are generally logical, all we get the approximation for this equation in terms of these growth rates. So simply get exponential growth, where the, where the exponential growth rate is. 15:29:51 Whoops. And so the statement that I want to make here, and I want to say it very rigorously but we've demonstrated we've sort of 15:29:59 delegated this thoroughly with computation is that if that this quantity of new A plus B ry is a good approximation for fitness. 15:30:01 And. 15:30:10 And so, they'll say if you do a competition experiment between two strains, two different phenotypes or two, or an R in type in a Nirvana type, this quantity, new a plus me rise is a bit discriminated between 15:30:29 this this Young Australian in this in these two random phenotypes. 15:30:47 Okay so without on the table. 15:30:50 I'd like to sort of proposal theory for what might be happening here. 15:30:53 And so there's sort of three comments this theory. 15:30:55 But the yellow strain in this case is better than the blue stream simulation of the civil unrest experiment using for equations at the approximate question, your screen. 15:30:58 One is that phenotypes are constrained by a sort of pH specific trade off carbon environments. 15:31:05 So simply saying that in the space of growth rates and I trade and trade possible phenotypes can exist in some region. 15:31:21 And that they're that they're sort of underlying this idea of the trade off curve is that there is some competition for intracellular resources. And, you know, some, some data point in part of this idea that there is some kind of trade off is that if 15:31:31 we do experiments on knockouts of Pseudomonas Aeruginosa where we essentially try and make Spain's which which cannot do the second step, reduction. 15:31:41 We see that the growth rate on nitrate increases blood relatives. 15:31:49 And then the fact is smaller but but still there, but if you reduce the capacity of the strength of a pain to do nitrate reduction of the growth rate, and I tried production. 15:31:57 So kind of 15:32:01 the second element of this theory is that the curve is concave down at Ph. and that sort of 15:32:08 implies that the fittest phenotype, is the solution to this sort of constrained optimization problem with optimizing fitness and as a constraint of this trade off curve in the fitness phenotype the fittest phenotype is going to be this generalist or nitrate 15:32:22 specialist. 15:32:25 Right. 15:32:25 Okay, and now the third element to introduce God is returning this question of how my Ph. impacts physiology. So, when we do experiments on our screens, which were isolated from neutral soils. 15:32:43 And we be measured their phenotypes in pH 7.3. 15:32:50 They're up here. So some modest values of a new a new I. And then when we, you know, type them at lower pH what we see is that the nitrate reduction rates, jumped on so harmed but when I tried reduction significantly. 15:33:05 Don't on so hard but the nitrate reduction significantly. Okay, again this is possibly having to do with the fact. And, you know, I'd like to discuss issues with this idea but this is possibly having to do with the fact that nitrate reduction, less cytoplasm 15:33:19 is buffer against 15:33:23 ok so the third element to this theory is that perhaps this trade off curve is deformed asymmetrically as pH decreases so that is to say, this is the fate of curve, relevant to ph 7.3, and then press the right after for PhDs point oh, and has decreased 15:33:39 more in the new iteration, and the consequence of that is that the fittest phenotype is now nitrate specialist. 15:33:49 And if you continue this, you can hear the form the curve asymmetrically in the right direction, what you see is the phenotype moving closer and closer to this new axis and the assumption here is that essentially that I talk to be harmed you I'm more 15:34:03 than the way. Yeah. Is this a theory of physiological trader, or does it allow for like ecological compensation like if you have multiple strains going at the same time. 15:34:15 Maybe they are fitter than what this would predict until the end is the input of this just, is it the physiology of one organism or yeah so so the statement that I'm making is about the physiology of one organism but there's another question that if you 15:34:30 say for example if you if you, if you generate phenotypes along this curve and you compete them all together in a simulation. 15:34:41 You is this is a strange still the fittest or is there some other combination of phenotypes that are fitter and the answer is that this approximation for fitness to hold them in that competitive. 15:34:56 So, this is saying essentially that the fittest physiology is fitness, because the idea that these points and there is. 15:35:08 And also that they are fitness in this, you know, highly competitive. 15:35:21 So I'm, I'm trying to understand the toxicity thing. Yeah, so maybe it's more of a comment but maybe I'll just say it out and you'll tell me if I'm if you think I'm right. 15:35:30 But, so, in order to, you must make nitrate, when you metabolize and nitrate so naive Lee. Seems like, that'll be a problem if the source of toxicity is is nitrate. 15:35:43 Yeah, but I think what you mean is that if you make nitrate, you can at least let the nitride go, I mean if you consume nitrate you can at least let them, but if you consume nitrate then you have to metabolize it It must be inside the cell or inside the 15:35:58 classroom at least in some sufficient concentration or for metabolism is that some like appropriate reading of what you're saying. Yeah. And if, and if you are a strain that utilizes nitrate that you're perhaps undergoing a greater specificity, as a result 15:36:12 of doing so because 15:36:16 that apparatus is localized localized. Awesome. 15:36:20 Yeah. 15:36:25 So this is a really interesting idea but I'm thinking a little bit about point one here. 15:36:31 This, this sort of code that you have, in principle, if, if you believe the regression coefficients that you have that you could then you could sort of approximate what the shape would look like by taking all to the 17 possible genotypes. 15:36:49 Would you not Does, does that look like this. 15:36:52 Now, so you're asking if I get all possible physiology is based on the regression, correct, like using the regression as a Ford Model for making physiology, needs to be at seven. 15:37:05 You could, in principle, approximate what this would look like right yeah that's an interesting point I am skeptical that that would would do anything like tracing out this curve. 15:37:22 I think 15:37:22 what yeah i mean i think i think that would probably be taking the model to literally as a, as a mechanistic generator physiologist. 15:37:36 But I think, to the broader question of how do you, how would one sort of try to characterize these trade off curse. 15:37:45 I think, you know, okay so so to characterize these hubs globally, maybe one needs a very detailed and accurate biophysical model. Okay. 15:37:58 I'm going to cut off questions so we don't run out of time. Okay. 15:37:59 Right. And then you can ask afterwards. 15:38:02 Okay, I think I'm mostly done. 15:38:06 Okay, so I just want emphasize here that you know this this point at which the fittest strain is is on the access corresponds to pathway splitting in the way that I'm talking about it. 15:38:18 So here, nor screen is preferred over in our Spain, so perhaps the pathways but 15:38:27 okay so as I said this is very much a work in progress. 15:38:32 We are working on his so you know this this experiment and thinking about it in terms of this proposed theory has revealed to us that you know Englishman experiments for be able to finish points on these trade off curse. 15:38:46 And so, warming English some experiments at lower pH is yet lower pH is where you might observe pathway splits to the fittest is necessary so we're working on that isolating spans from these experiments, you know typing, rather than like I'm rushing and 15:39:06 these trade off curves which we believe that we can do vocal sense, around this finished point, working on, and this is something that you post uptown Crocker who have met, I'm thinking 15:39:22 okay so that's yeah that's basically it. So, you know, in summary, we observed in this management mix they do the pH drops variation in this marriage, you know, as mentioned on the structure and what we observe is consistent with a half a spring but tentative. 15:39:41 tentative. But are we, you know, from, from what what we understand about how pH impacts it in education. 15:39:52 It's possible that pH is causing this nutrient toxicity which is somehow driving and be have the sort of proposed theory for how pathway spinning occurs, and it's by this by this asymmetric trade off this space of these two eating processes. 15:40:12 And with that, I'd like to thank my great mentors. 15:40:17 Their mother. And also, folks who are working with cracker. 15:40:24 Thanks. 15:40:46 Yeah, maybe they only reveal the fittest occupied points on the trade off curve, based on whatever was in your sample. That's right I was sort of curious, are you, one can also imagine creating the split pathway genetically that's tractable underneath 15:41:02 the eye slits yes had is that in the plans to, it's not directly in the plans but I think we are proposing interesting so what you're saying what one might do is start with an already very fit for the natural fire, and then knockout night fight reduction. 15:41:19 Right. And nitrate reduction in your model sort of predicts that at a low enough Ph. You get that result yeah i think i think it's interesting question. 15:41:29 Based on, you know what, what, Terry was leading to the beginning where there's very different taxa, which are responsible for which which can grow at low pH. 15:41:35 I kind of wonder whether a phenotype that we isolate at a higher pH is would be capable of growing up, or peach at all and so perhaps that approach wouldn't. 15:41:46 I said okay but maybe we're asking is, if we take this strain and makes it harder knockouts and throw them in at pH six, perhaps that for example yeah it would be out that would seem like those knockouts already existed from one of the intermediate slides 15:42:01 right you had those two knockouts that prove the trade off in the first place. So those were for EA one cinema storage NASA. So I think that that strain is probably less guys that would isolate from here. 15:42:14 So, But I, from what I understand the it It's out of the question. These days to do those knockouts on the CD minus. 15:42:25 Minus very strange. 15:42:27 Yeah. 15:42:33 I think one of the most underrated environmental factors that then when you're not keeping track of it difficult to get hold of it is not so much the pH but the buffer capacity. 15:42:46 It's right it's a low power capacity allow them to produce a change in pH. 15:42:53 And I mean that that is just so important. I don't know how to get that. Yes. So, are you arguing that in the management I'll make a note we're not keeping track the buffering capacity and that could be different, different. 15:43:11 Sorry, can you just clarify what you mean the buffering capacity of the ability of the cell. 15:43:21 I see I see I see you're referring to the buffering capacity of the natural environment, not intracellular buffering capacity. 15:43:35 Yeah, you're not also saying that one should be looking at the pH dynamically during the enrichment experiment right 15:43:47 in the situation was low, low buffering capacity, I say that because, you know, experiencing pH changes and tidal wave there's no time to respond. 15:43:58 All right, let's think carnal again.