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informatics drug phosphorus metabolic chlorine Prospektion chemical molecule Magnesium Potassium cell protein uracil level atom biochemistry processes chemical element organizations nitrogen DNA sulfur synthetic biology carbon Wasserstoff coli Iron chemical structures polymer oxygen DNA replication form chemist selective
type unsaturated Trübung affinity growth media chemical Prospektion synergy molecule machine uracil Library DNA and RNA coil organizations steps Polyester sterile media firm death systems RNA cultivation coli compounds monomer polymer turbulence Oast house Spektroelektrochemie form selective sense Blood vessel Twins density Polyamid Nutrition Morphine chamber case formal man Trihalomethane strains analogue Natriumhydroxid Methane cell overexpression molar level natural selection Schmerzschwelle composition asset DNA Teflon pressure antibiotics molecular biology flow conditions metabolic pathway variants electron transfer chemical structures substrates basic separation chemist Crystallographic
Frontlader natural selection type machine fluids secret sterile chamber beweglicher
near Butanol unsaturated density Bohr growth media Primärer Sektor kinetics set case food man strains hard machine race stress P sites tolerance uracil level processes natural selection Butanol composition oil concentration Stickstofffixierung Chemical oscillation mutation water stem compounds turbulence bottles resistance selective
Digital elevation model Bohr growth media operation guanine chloride cytosines set chlorine formulation Alpha enzymes machine race protein uracil coli Cycloalkane Methylmalonyl-CoA-Mutase biochemistry organizations Mitochondrium protein synthesis rates steps genome eukaryotes polypeptide Methionine media firm systems Mühle van Säuren Mast cell RNA cell Brom redox potentials bottles uracil methanol biosynthesis sodium hydride Deoxyribose species factors polypeptide chain RNA deformation case genetic information DNA man strains Methane cell control Thymine translation Harnstoff chemical formulae Färben slight triphosphate composition Wasserscheide tRNA precursor DNA formal pyrimidines mutation initial function Pasteurisieren Thymine basic
Digestion activities Trübung density growth media gene traces relaxation chemical man strains originators chromosome machine hydrolytic uracil modifications processes type delta Maische tRNA DNA Chloral genome solutions charge mutation Tee Drops Säuren cultivation coli remove chemical structures Thymine base pair sequence selective
metabolic guanine cytosines Adenine chlorine chemical strains Strength Thymine poison natural selection type triphosphate genetic code Plate guanine man cytosines antibiotics systems chromosome Säuren Song of Songs Kanamycin coli Thymine basic Polyethylenterephthalate stuff
type species operation synthetic biology set additional pressure strains branches conditions artificial cake temperatures Oceans control amino acids species base pair sequence
I was able to gather some of the PowerPoint pictures that were needed to tell you about our exploration of say no DNA in vivo using clora uracil so I apologize because this PowerPoint file has been going through different computers and I am not feel that let's say the quality of the pictures and everything is been a concern throughout this process okay this is about navigation this is a metaphor that we use with the public but we find it pretty accurate so to speak we are having a evolutionary trajectory trajectories but not so much in the second space you probably remember the sequence space that was introduced by john maynard smith long ago where he described the evolution of organisms as trajectories in the combinatorial space of second seas of a tgc or proteins with a wider alphabet and so on now the very basic structure of living organism is chemistry as a we had a wonderful examples of what what this elaboration chemical elaboration where it leads it is leading us in terms of having new expanding the alphabets and so on now the chemical space of off life uses a limited number of elements for instance the element chlorine is not well it's not used in the biochemistry of most organisms the element chlorine is used by certain in the secondary metabolism for making drugs and there are many chlorinated molecules but it is not used in the making of let's say e coli not at all there is ZERO chlorine atom needed for making an equalizer of course carbon hydrogen oxygen nitrogen phosphorus sulfur magnesium potassium and so on they these elements participating in the construction of the cell and they are required by every organism that we know but for instance iron is not used by by some lacto bacteria so there is some something that was that people have for quite quite some time already wondered about how to cross meander AF and Darwin in some way and what I'm going to to to to to talk about is a very simple pedestrian approach to trying to change this alphabet using techniques selection techniques which are which have proven reliable energy energetic slab but which have been automated at least that at the level required for conducting experiments let's say perhaps not forever but for an indefinitely long time well I'll just I just wanted to mention that we use on now the the term xenobiology to to to define this sub field of synthetic biology xenobiology was a word that was used together with EXO biology and so on so there are too many words used for talking about alien life not necessarily extraterrestrial but life as we don't know it so we just you know hijacked the xenobiology and say nobody is about living organisms chemically deviant from terrestrial life as we know it whether it is extraterrestrial or made here using genetic techniques or chemists organically strands phone okay so just a very very yes simple metaphor as I said is to consider possible let's say if we ask ourselves what is able to proliferate to replicate chemical II so it might be a very vast and actually experiments and that you are going to hear about just after my talk buyer by Phil holliger is going to convince you that the are going to convince you that the ranch the scope the very the diversity the chemical diversity of polymers able to project is probably very very fast actually so it says it's not as a small just a small part of of molecules soul now life as we know it on earth you don't know it elsewhere so far right has been it's a sub part of this and now we could consider virtually molecules that we could conceive or access to samee empirically and so on and we added and now thinner biology is about navigating this so as to start from a living bug that is made following canonical rules and to evolve it because we we are we are probably unable so far to design forms of life completely different chemical er very different chemical e so our best trick is to use Darwinian evolution to move away from mother nature to somewhere else I will not argue too much about the reasons to do that I mean extra scientific reasons to do that like protecting the younger or not and so on because we have written on it many times so unless there are questions I will try to avoid this
another way of saying it is defying xenobiology that it's a it's a kind of an expression technique just like astronautics is so astronautics is absolutely as a challenge astronomy at astronomy the science of astronomy was needed to do to lounge artificial satellites and so on and we see that there is that there is a synergy between the two but politics is definitely not a tsunami and it's a technology if you want or whatever xenobiology in the same sense it's an exploration that is not reducible to biog as we understand biology it's not just a matter of giving an account of life it is how to explore all the forms of life just like ok so among the different very briefly and in just a cartoon how to change chemically life analysis suppose that and this little m air is a monomer embedded in a polymer that has been condensed by the biosynthetic machinery so long together we could we could consider a full replacement I'm going to talk about this later full replacement of this monomer by something we are an asset okay we could have also something else which will which could be addition of monomers and this is exactly what we heard yesterday in Floyds a talk how to introduce new new monomers to be condensed together in the same polymers as the ones which already exist and we have to consider a third way of challenging life chemically which would be two condoms to polymerize these additional monomers in a sec in a topologically separated class of molecules and that's with Pete and that's what we call the same nucleic acid and so on in the case of nucleic acids and JSON chin by specializing ribosome is attempting to do that in a if the polyamide or polyester world whatever okay so these are basically the the the three ways of altering morphine morphine chemically life so to speak now it should be realized that these experiments experiments attempting to change to substitute to replace a monomer started very early on and we're considered to be curiosity to laboratoire and actually the first successful attempt was done in 1957 just you know at the birth of a molecular biology by Jove coil in other Pasteur Institute where he gave saris it's really am sorry sellin cellino methylene to an e coli strain requiring methane and that bug that strain unable to make methylene could grow with 70 me funny so it was considered completely a futile in a way then it proved useful for phasing in crystallography and so on and presumably this orgasm the sorrento methylene ecoli was the first historically was the first say no by aunt the first whatever we we named it and we saw our our project was to attempt such a challenge but at a deeper level and so DNA and the the chemical composition of DNA was attractive why because there was already a vast body of knowledge coming from a pharmacy and so on showing that many many molecules especially nuclear bases and so on many antibiotics we're sort of fooling a fooling around with the DNA RNA structure and the and the monomers of them so we resorted to okay now so before I get in the deed in the depth of the chloral uracil genetic setting let me just I would like to talk about the technique because suppose you have some usage of an assets often of an monomer analog or a non-natural chemical venue in order to to see these use of such a compound be improved by natural selection we must be able to maintain a population of sales for an indefinitely long time and so that was actually the main difficulty in in this project because this was a problem supposed to be solved and actually it was not and the reason why it was not is the following it is that if you conduct well this is absolutely unreadable I'm sorry this is PC to make interest me to a PC to apple so let's say the chemostat has been a technique to it's a it's a cultivation technique in which there is constant volume there is a feeling at a constant rate and so the theory is that cells will grow exactly of the delusion right and varies in under under these conditions there is a limiting nutrient and actually everyone is expected to favor selection is should should should should be for organisms with a higher affinity for the limiting substrate okay so are we interested really in having high affinity for a limiting substrate well not so much what we would like to have is better it's a it's something like a system in which selection would be for the fast for
faster growth even at saturation of all substrate but we would like to have organisms growing at an ever higher right and the this in in principle can be established in the Derby does that so in a turbulence that everything is in excess and it's like if you want there is a little sensing a sensor recording the density of cells and there is let's say a valve that opens and refresh renew the medium when give a certain threshold is reached okay so in principle this was a invented a long time ago I mean in the forties right I think it was a some guy boots all or Institute us out somewhere it was a clever but lazy technician and he was tired of coming and weekends to refresh LJ growing with light and he had the idea since there was light to put a you know a photocell at the back and so when the Dead City was I enough you know it would it would have the the valve are renewing the medium so they made of paper in science it was called the turbid or stat so the principle and physicists at the time you know they were going from the Manhattan Project to a biology and they found that it was a super system and so on analytical it was very nice and so on but it did not work and the reason why it not work is I'm sorry to say that but this is something that I just carry you know just share if it is you know structure and we are not biofilms and so on but actually it's a drag in evolution so biofilm a biofilm it's also called the world growth actually if you set up if you set up a device in which you have a constant flow of excess nutrients you might say that you have established you might believe that you have established a right queen microcosm in which you know the faster you run you have to run ever faster to stew to remain at the same place all right that's exactly what we want as evolutionist can we call it will finish whatever well actually there are many other ways you know this is just wishful thinking if you remain the same in the same vessel because there is a much easier way to stay in it is just too attached to stick to the walls in this case the nutrient flow will just help you sticking a ver bad I've ever better okay so so this was a difficult technologically challenging problem to solve for the reason that Darwinian selection was precisely selecting what we did not want so and this problem had been had been recognized not properly interpreter stood but recognized early on as soon as people set up a torpedo start with E coli and so on they would see a world growth dental plague so to speak in a cultivation vessels and in e.coli in which medium it takes three days plus or minus three days so people at the idea of making a teflon vessels and then it will take 20 days you know but you will get teflon stickers you could create disease or whatever other so it had to be solved algorithmically so to speak and we were not engineers and so on and we we had to to to tackle this with Rupert and the way we we managed to solve this was to make a twin system so suppose this is a turbid oast yet you know so this is a closed valve this is an open valve so there is gas flowing here there is a nice siphon so that the liquid goes here and pressure equilibrates here and all this goes to waste liquid and gas now in a turbid or stat I are victorious at exist so there will be measurement of the density and presumably and this valve would open and close according to the density of conditionally to the density of the bugs okay so there you can have you know lazy you know stickers and so on and you can have good guys free-floating planktonic they say all the same alright but now suppose that the status of these valves challenge so that this happens is that you get this pattern you know close close close close close close close open and so on so this was fluidics right so it's establishes now a path for the fluids so that only pressure can be only a released here so in this case this will amount to transferring the content of the left chamber to the right now this is simple plumber right but when you have done that you have you have separated the good guys who have been floating here from the bad guys who like to stick and now suppose that the fluidic configurations changes again and that deadly says sodium hydroxide 5 molar gets in okay so in this guy will you'd say genetically that well the bad guys will have no progeny and any they will not have any descendants in the rest of the process okay so I make it simple but this is a like a little washing machine you have something like 30 or 40 steps and the very top og of the whole device is configured so as to make sure that no no refuge whatever that took some time exists so that there would be a way of escaping certain death if you stick as soon as we could so then yes to go to go fast in this so in this case after rinsing and so on it's like you know I do tell you get the chamber that has been re gone back transfer purging the other health and as soon as we could
establish this cycle which is a
summarized here you know going from culture going from left to right purging rinsing and so on but back transfer and purging of the other health as soon as we could establish that we thought Darwinian selection in action and something very simple that people had never seen because of the biofilm thing okay and we could run it was some time ago we could run the torpedoes that for the first time it was in 1998 so the way
the machine looks now it's in a there are many different formats but is it a standalone that we are that we leads to the industry so it's a standalone you know it's a pretty robust thing and you can unplug it put it in the elevator replug it and so on and it uses it has the only the only entities in motions are fluids up with and valve openings and so on we don't show there is no close-up because there are some secrets for having this airlift chambers go running forever but this is super robust and this has been a taken care of by engineers notes of its very robust now
there are two ways of there are two ways of using such a device navigation device so to speak one is turbid as that as I mentioned earlier and in this case what will happen that suppose there is a clock so every five-minute it would be time to get a pulse of food or no or nothing depending on the density of the cursor so see if density is too high then you get a pulse of constant a pulse of constant volume in a culture at constant volume and if you do that every five minutes you will establish exponential delusion so exponential delusion is another a way of expressing it is you impose ascertain ascertain division time generation time so in the turbulence that you would say if you if the density is the culture calls for a pulse every five minutes that would establish a generation time off I don't know ten minutes or something something that is far too short okay so what will happen is that the culture will adjust to have delusion exactly at the maximum growth right so those mutants which have the smallest generation time will be winning the race so we inner turbulence that you establish this Red Queen world and since we are purging there is a cycle of purging that is overlapping this cycle of refreshing the medium we add this time evolutionary this evolutionary machine good now there is something else that you can do with the machine lagnachi that instead of deleting too much and trying to attempting to to to to to to sector culture at the maximum at its maximum right of delusion and there there by establishing the the lowest generation time there is something of that you can do which is to send a purse that would establish of a certain volume that would establish a generation time a reasonable one let's say four hours but then what would change depending on the density the density of the culture would be the composition the composition of the medium so in a case ID so if things go too well densities is high then a pulse would be sent of stressing and stressing a pulse of stressing medium would be sent to the culture and if things go too badly the density if culture gets several pulse in a row like this the density will drop remember that we are now in a concentration and then at some point density will be too low and then the the device will send a pulse of relaxing medium so we will see later that if we have five min ear and Laura uracil here we could let the culture decide exactly the amount of stress it wants to get okay so the first the way we applied it the first time is to
demonstrate the the potency of a such a simple technique is to improve the resistance of e.coli to a nasty nasty compound which is called n-butanol at the time people wanted to increase the the right of production of a beaut on all to make fuels and so on and this is the kinetics every scenario kinetics in days you know you have from the 0 to 300 nearly a year off exactly what I said it that the number of pulses with medium containing butanol and so there are two bottles one one bottle with the butanol at near saturation and the other bottle or a relaxing bottle no butanol stressing bottle plenty of butanol and here you have overtime all the other the response of the culture calling for ever more butanol containing million and after let's say about four month or so a very high level of resistance is reached now there would be we don't know of any other technique you know engineering or whatever for saying you know I'm going to change the to direct to introduce mutations in a completely irrational way so as to get this kind of tolerance because our knowledge in a biology is completely insufficient but now in a I think it was called selection is a kind of irrational design you know as I think it was brainer who said that and the so by by resorting to techniques like that if they are reliable we can we can perhaps define but attack strategy in which we design something at the beginning but we don't have to design everything the evolution and all the challenges that would be required to get to a certain adaptation we just have to make sure that the strain can no longer can no longer get back to a given setting and an let natural selection do the rest ok so that was demonstrated for solvents and believe me these are very hard hard adaptive processes because butanol can get everywhere you know it's a it likes to to go in an oil and water at the same time it's it can go everywhere in ribosomes and so on so many many mutations and actually it was a mutator strains and you get android's of mutations fixed if you do that okay
so now it's time to adjust well I think I'll be brief on this so you know that will not go away from the the natural world the the first attempt that we made with this morphing the chemical constitution was to alter from the protein priming because there is a big divide among species there is something that it's all so rule that nobody already understand why it's like that but it is done exactly that you know in a in the in the branching of eukaryotes archaea and bacteria bacteria don't use methionine to prime protein synthesis they use formula methylene and there is a special enzyme that exists for modifying methanol tRNA and there is another enzyme after translation for removing the Fermi group now in a RK and eukaryotes there is no such for me group so the priming is done directly with methanol initiator tRNA and it is supposed to have been sold since you know three and half billion years okay so you have completely different organisms mitochondria which are the bacteria which have evolved within a eukaryotic aust and as well as a chloroplast do from me late methylene for priming protein biosynthesis so a simple question was suppose I get rid off methanol methanol GNA initiator for mill methanol to initiator ribosome and so on the big machinery gets to from in methanol a peptide the day formulations I'm releases the formal group methanol polypeptide is further processed in for most proteins releasing methane in that can fire a cycle again and and this is for bacteria and energy there is just these steps for me transferase and the formula the deformities don't exist and they are nicely set in an operand so if you remove the operon from e.coli you get this lifestyle so what happens when you do that first well when you do that egalité doesn't die but let's say it fit its Fitness goes down well it's not a slight epsilon right because the growth rate dropped by a factor of four it means that in nature if any ecoli bug and it must happen every day in the gutter as the bad idea of losing it's a formulation system well let's say it will not win the evolutionary race right now if we take this cripple which is viable nevertheless and we put it in the machine then we can test the scenario that mother nature let it doesn't let equal i do on its own and so we don't know what it would be and so the question was how long will it take to get rid of a trait that equal i has been entertaining or its ancestors of entertaining for three and a half billion years well 1 million year answer is biblical it takes 40 days and 40 nights all right and actually what you see here and we are very proud of that is that these little shoulders here each of these shoulders its analytical it's it's beautiful each of this little shoulder is an adaptive mutation so actually you of the order of 8 to 12 mutations are needed to recover to visit or resurgence it's a good resurgent it's a good word to get back to well nearly normal life and so there is a paradox here okay because there is the products when I tell you that for there is no exception nearly no exception for formulation of translate in translation and there is no and it takes just a little plomberie okay add a 4 and a month to get rid of this straight they should be everywhere so presumably it is the competition among peers right that prevents evolution of formerly free organisms well so when we saw that we we can really where we were telling ourselves we are not asking enough you know we should go in the very depth of of deformation of this and let's get to DNA all right i'm sparing you what happens when i'm sorry sparing you what happens when you put mutators and so on we have done the experiments many times and i should say also that when you look for the Mutai adaptive mutations whether in the genomes guess what you are you find you find for a ribosomal protein and the methanol generation today's those enzymes involved in the cycle i showed earlier okay but let's get to you know let's try to go to something harder to do okay what would be really hard without
would be to change the base of janae because a DNA has been around we don't know at least since Luca also three an alpha billion years and there is absolutely no exception known to the composition of any cell as far as DNA is concern you always polymerize cellular DNA in all possible species in all species that have been analyzed from the triphosphates the deoxyribose I triphosphates of adenine cytosine guanine and thymine okay so since chloro you are silly now the case of thymine is interesting because it is the it is the the component that is different from RNA you know in RNA you have uracil and in thymine you have in DNA you have five min so this is in a way disentangled the five min biosynthesis disentangled from the bio synthesis of RNA precursors and it is a easier to to to to put under strict nutritional control so when phi a the enzyme that methylates uracil in 25 min in the DNA in the dania precursors is deleted disrupted then you can grow e.coli nevertheless by providing it with the base thymine or the deoxyribose ID and there are enzymes phosphorylating it and get into DT MP and so on so that DNA is made with the proper components now as I mentioned earlier 5 illusional pyrimidines had been described early on and shown to incorporate a little bit in a in DNA of mast cells and it was not clear whether it was a there was it was a killing cells or not but there were some in that it they might survive invasion with the widow let's say broad bromo uracil and get back to normal life okay so as Pete mentioned yesterday so there is something actually we so we could choose between different and we different SS like this and we we went from Cloral from chlorine instead of bromine or whatever for the reason that you cannot the redox potential is not appropriate for just having chloride being a cleaved off here also it's more floor Furion would be too ambiguous it's it's a base that is very ambiguous it pairs with with G as well as a and chlorine so the chloro your acid was good because it's it's a kind of yeah it was right a borderline you know pairing this G but not too much so that genetic information could probe presumably be propagated using chloro uracil instead of thymine but it should make a lot of mutations and actually that proved to be true as you are going to hear in just a minute ok so the setting you can yeah this is just showing that a glory you are sealed paired with G is a either is more favorable than the GT corresponding GT pair ok i spare you the metabolic setting just mentioning that we are just at the beginning it was not very difficult I mean something like 6 Jenny genetic changes were needed to make this strain able to use either chloral uracil off I mean and we had to mobilize we mobilize the so called deoxyribose will transfer is from lacto bacteria and all this construction work was done by Madeline boozing at at Sarah okay now you remember what I heard earlier you have heaven and hell relaxing medium and stressing medium so two bottles one with chloride uracil one with thymine and on you go right so we set up this device now there is us with that so that this is time I'm sorry it's a so it's a it's not it's hard to read probably from so they 01 day 145 you know and this is the the plot of non Simon containing medium cold by this e coli culture and as you you see well equal I the e coli culture was calling for evermore medium not containing Simon until it reached M&C patient from Simon at after about five month or so we did it several times I've actually it was pretty reproducible first thing that we did before getting you know Jake analysis of this yeah this
is you too so that we can some parameter at the time that was imposed at generation Tanis on but it was it was it was pretty reproducible which will be so then we analyze them sorry it's not good but we went to of course we did chemical analysis of generally digest hydrolysates of DNA and we found Cloral uracil but we also found that even when thymine was no longer present in the Natori nutrient medium that some thymine in the bugs after two thousand generations still required still made some time in even though we are disrupted Phineas in tourism actually we could we could we could we could figure out what was going on it happens that in trna is there is a so-called t loop and there is a post-transcriptional modification of some uracil for making tea it's called the till you because there is a ribose if I mean and we don't know those bugs manage but presumably they are able to scavenge to retrieve the tea that is made just for make you know few ties cycles or whatever with Jana just to grab to grab the traces of tea well so the solution was a simple third surgery and the removal of the gene in charge of modification of that you're ISIL and as soon as we did that we did that in the strain we saw the little remaining peak of thymine disappearing so it was the x-men so of course in the following runs from the start from the background the chassis we removed those methods and jeans so as to leave to Eagle Eye no option okay how perfect thank you okay so I'm yeah so well I should say that so we got to this phenotype you know so that was the original strain it required five min to grow did not grow with glory Rasul and after 145 days it grow with chloral you are still alone we finally grew it still grew pretty well but not as well as with Cloris ill when for for our generation time was imposed during the VR active process we got good growth with both okay and when we remove the trm a that I was a the modification gene for trna it was same phenotype okay well
there were pretty we were struck by the fact that there were many mutations and mutations of a type that is not that is not the most frequent in equalized a 2g or 82 GC mutations well I'm pretty you know for for 1,000 generations you know 1,000 about 1000 mutations accumulated in during the adaptive process and we got also a mutation in the mute l which is a mutator gene so things that are observed commonly when you calculate equal I for a long time mutator jeans like that pop up okay so that was the array our rehearsal run right this this is this was published dinner sometime ago Wow now this is okay so up to it well if you see the curve that's that's okay i'm going to tell you the X what is in the X and Y axis so these are these are timing days these another run but it was done in the following way so we said okay we learn from these first experiments that we have to remove tRNA modification gene and so on so we start again but now we are going to let the machine takes care of the following it will adaptec adapt by swapping medium between relaxing and stressing adapt this equalized train to two of a pure pure Clearasil nutrient medium and then once it has reached complete replacement of Chloe with chloral uracil then let's accelerate it introduced at forever and now we are at 9,000 now over twelve thousand generations but the results i'm going to show you are we the the genetic analysis of bugs at about nine thousand generations and so this shows all the generation time drops and actually it sort of stabilized around two hours we don't know but evolution is still going on because mutations are accumulating and equal I would grow at about one hour equal I wild-type with a Simon about there but this exponent way we are not going to stop so because we would like to see how far this trip this replacement and adaptive mission mutations can offer it will go so this is there was a parallel run completely independent from the same ancestral strain so to speak the same chassis we evolved it as a long time and we got a very similar very similar process under to as we speak these two cultures are calling from are accelerating in turbid instead ok now the genomes were a sequence and we saw an awful lot of musicians so here is a plot of for the two cultures the number of mutation that I've accumulated so there are in the e coli genome there are two million to two million over 2 million 80 pairs had to about 22 million GC pairs five about four and four million and five hundred thousand base pairs and in a matter of over a year of cultivation now we have accumulated something like 8,000 8,000 mutations in this gene ohms the venture I mean the various mutations when four base pairs are three possible point mutations right either a to see a 2g a tutti and so on and in a majority these mutations where a 2g and a2g means that it is that the ambiguity of chloral uracil leads to a mutation of an 80 pair by being in the template and pairing ask Laura you with in glory in the template and having a deed GTP incorporating in response to it this trend so this is measured here we just measured the number of 80 pairs that disappear so minus 200-400 minus 6 a nun and culture the first culture you see that there is a drop and it is slowly going to GC enrichment and in by the androids and for a reason that we don't really understand yet the second culture it at the same trend during the adaptation to Cloral uracil but once we give we gave it only chloral uracil and it was interviews that then the trend reverse now it's it's a GC 280 not 82 GC even though the mute the same we there is no easy explanation for this fact but we let it go we'll see where it will and now well I'm sorry so for the culture the first structure so there are nine thousand and no I'm sorry nearly 8,000 mutations here and fix thousand mutation in the second culture and there are 142 mutations which are exactly identical this is more than you would expect by just throwing mutations you know and picking at random but this would be counting without selection so if some loss I are under selection for adapting to Cloral your acid you should expect them proved yet also them there could be odd spot and so on so we this is what was interesting also is that there are so many mutations accumulating is that by just plotting along the chromosome where are mutations and the type of mutations appearing so a difference between the skew there is a clear skew showing where the replication origin is you know so if we are not known whether replication origin was you know just by following the density of different types of mutations the skew is absolutely abusing the two cultures you can actually see
this ok now after six South thousand
generation so from time at irregular intervals what we did was taking isolates from this culture there remember that they are always growing in competition and adapting to Cloral your acid introduced at and from time to time we take an isolate we spread it in a plate and we we in display there is some chloro urs e to grow but then we can ask whether whether it is still able to take a thymine and or not and so progressively we saw that Phi mean was less and less palatable to these trains until this very creative stage stage where we add you know this is a one isolate has been spread on the plate a little well has been done and thymine is introducing the plate and you see that time in now is a poison to this strength I'm sorry not sure this is just a trick we got great and plate so there is plenty of it here and it's like you how you take antibiotics and stuff but now yeah sure sure yeah yeah with pleasure man but this is exactly exactly the pattern that you would get with kanamycin or an antibiotic so and so this is sort of this this is telling us that xenobiology is not going to be too demanding intellectual you know because we just by letting bugs adapt to chemicals if these chemicals are acceptable at the beginning natural selection we just make a noise like a noise like we just make metabolic systems forget about the canonical components i think i'm going to do end up here and the two well i think Pete talked about that yesterday about the what we are doing because Gloria you're a CEO of course it's just the first stage okay so the next stage that we are going to to have other basis get in and we Valerie pezzo is constructing the strains it's more complicated because we have to get free to get rid of nucleotide ready tales and songs of metabolic resetting is more complicated but now so the game is in lovin you know they are rehearsing and investing what different types of bases could work in PCR instead of canonical components and pete has made a really incredible effort at making all these triphosphate and testing them in pcr and now we are with the system so glory or are still safe euro cytosine da za adenine and d is a guanine work we're in pcr so they are the these guys are a dollar the next candidate for invading coli okay yeah no
let me finish with this if it is readable yes because yesterday during the discussion there was you know there is well that's a joke runs like yeah everybody's like God you know everybody has its own definition of synthetic biology mine is it is I would say it is the experimental branch of metaphysics you know for the following reasons we cannot say at the same time that we are omnipotent you know that we can do anything and that what we do is innocuous to the younger or not yeah you have to you cannot have your cake and eat it you cannot say we can do anything transform the living world and so on and that will pose no danger to nature to natural environment on because species are tinkering and so on already okay so we have to choose so but we should have a way of measuring whether we are really omnipotent omniscient who we will never be but omnipotent we might become of me but and that's already a big threat for the environment no I would we would we a monitor that just measure it so I refer here to a well-known idea in a computer science it's called the Turing test you know how do we know that the computer of things well it's hard to tell so the best way is to have an operational test you know and submit to a panel of people answers from either human or a computer and say well if the panel of decides that cannot make a difference between a human and a computer that day when we will be authorized to save at a computer of things right we could play we could we could adopt the same operational sort of Turing test for evolution you know suppose I don't know what you are going how you are going to challenge you to challenge a bug but let's say I've made in my back a bug that I have transformed perhaps I've change the set of introduced additional base pairs and so on yeah this is my business right you don't the only thing that you don't that you are not authorized to do is to sequence it otherwise that would be too difficult so you cannot know whether it's artificial or not now I give you this bug i say i don't know what you are going to do it you are going to change it for high pressure temperature and so on the Turing test would say the day a strain that has been artificially a reprogrammed bits the wild type in the same challenge I have understood evolution so long as I'm not able to beat nature have not understood able different you see what I mean and so the three outcomes from for experience like this going from wild-type to some irreversible state and letting it relax under control conditions there are three possible outcomes one is to have downgraded variance so that they will never recover while type while type fitness these are probably not the most interesting because it would be poor competitors you'll have liking the formal thing you know it seems to be you you can change the trait and then you get to the same fitness but of course the most interesting scientifically would be an upgraded development okay with additional amino acids or whatever but of course this if we attempt to do things like that they should be completely contained you know not originally or otherwise and if we do not manage for containing them they will spread in the oceans and so on so the issue is really serious so if we say synthetic biology is going to upgrade the living world we have to really master and control their proliferation thank you for your attention