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Romesberg and Herdewijn: Discussion

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Romesberg and Herdewijn: Discussion
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Bruchverhalten physical chemist transport nuclease cytosines chemical Materials International Space Station Experiment molecule Verzerrungen chromosome enzymes Strength race P sites uracil river period DNA and RNA accelerator pilot organizations oil synthetic biology abnormalities chemische Reaktion end systems toxicity GFP artificial Ionenbeweglichkeit RNA scale hydrophobic helices Spektroelektrochemie form sequence hopes stuff sense damage Überexpression Chromstahl case methyl Artifact (archaeology) specific strains analogue subject deamination terminal cell transcription Redoxreaktion Doppelhelix active site board cell nuclei storage level Butcher spontaneously processes type areas power molecular genetic code balance tRNA DNA proton mutation water DNA Repair metabolic pathway function chemical structures basic DNA replication base pair mRNA
growth burnings media enzymes machine electron P sites protein Horizontaler Gentransfer modifications vinyl antibodies eukaryotes firm systems packing Säuren oxygen hopes sequence Stop severe drug activities case Protein Sequence reactive restriction analogue Quantum entanglement Amber Methane cell transcription amino acids parents active site Stream bed translation Butcher conjugate Halogene type nitrogen genetic code tRNA pyrimidines DNA Histone mutation agricultural conditions function chemical structures pore separation Farmer basic base pair
purification metals organizations drug polypeptide chain steps bind Katalase additional solutions chemische Reaktion case chemical emission systems enzymes Säuren protein transcription color amino acids active site biotechnology poison
okay what sir its place ready it's half-past five so continue with the next bits on top of discussion I like this morning I think it's a good idea to start with the motivation of the more technical questions that my address not just speak up seeing specifically and
that we don't discuss briefly discussed soft soft the broader issues editor arise when we discuss something anomalous new genetic code systems I would like to point out that actually tomorrow morning there will be also are maybe more talks on low on related issues so we can if you don't have to finish this discussion tonight so does anyone have a more technical question for one of the speakership this afternoon of course we would like to know more about this transcription financial transcription so that was a teaser that you can you tell us more Floyd about so what kind of the molecules are feelings so um and we transfer transcribed um gfp to 12 EP three different codons to look at early middle late and we are only now beginning to look at sequence biases so we haven't heavily done that yet um we just got our own LCM SMS so we're setting up to do rigorous fidelity essays because I mean enemies been great but we just can't send we can't use them in the developmental fashion because turns out they have other things to do um but so right now everything is based on the buy it and shift and it's not a very quantitative measure so we don't really care about that yet except looking for differences what i can tell you i alluded to this I think is the tRNAs transcribe better and we attribute that usually t7 RNA p is resistant to row determine determine row termination because it's asked but we suspect that maybe running across transcription of our guy is slows it down a little bit and so structure prevents row determination so um I alluded to this to you as well I'm giving them I hope none of your reviewers on mines papers you'll be more um we've worked hard to get ecoli rnap work and it doesn't but there are aunty termination systems which we can deploy which turn it on um so that's right so you had a transporter to bring in here so not impossible yeah I've put you in my home and you said that imitated it to reduce the toxicity do you know how that makes me work I'm a transporter person so I feel like to know yes can you tell me we're publishing that I so i apologize i can't because this some of this work is funded by small biotech company and they get hyper sensitive to things like that the camera so two questions first one is you didn't say whether the cells turned green and the second one is what happens when you put consecutive unnatural basis um template so they don't turn green because we're in no case have we transcribe both the trna and mrna but what i will tell you in getting to that we've isolated mrna and the majority of its full length but of course i'm like unlike replication you get some that are bored of early and so we get something to do a board at the unnatural base pair and what's really interesting is they don't abort their on the board eleven nucleotides past reproducibly and that's a footprint of a ribosome and other people have already shown that and we've gone in with specific nucleases to digest them in the u.s. certain pattern and it is certainly that and so all i can tell you is that the RNA is spewing out of out of t7 and the ribosomes are izing it and they're stalling yeah and so the hope is that once we do it together the second question we have pcr-amplified contiguous natural base burst um it's certainly more difficult and I liked it I liked to be clear about this um we are not creating an orthogonal system our base pairs require the context of a natural system we idly we've never even tried and I don't think they would even form duplexes if they were all our hydrophobic analyzed um having said that we will create more codons than could ever be used if we never put them anywhere near each other and that's the goal it's not in any way to imitate nature to make our analogs as good as gcat it's simply to get to the spot we can stay please store and retrieve and evolve increased information it's a again from Floyd of anyone actually first ascent comment so one of the beauties of our natural systems that evolved a lot of very sophisticated DNA repair systems including base excision repair which deals with you know spontaneous deamination of cytosine uracil you know spontaneous loss of base where you end up with a key or any K pyramid Inc sites etc etc plus all the redox perturbations on the DNA itself and all sorts of other chemical damage that occurs so I mean your natural systems are highly evolved to deal with that and obviously keep the keep the code completely faithful and record replicable between subsequent generations so how is the synthetic base going to cope with normal chemical you know motivations and how will the natural system deal with that for example if you have a synthetic base next to say a uracil that becomes a cytosine that becomes dominated on the Earth's of removed to you end up with an AP site and you know it's all right i mean do you have any information on how that might work or have you really done that i don't know so I think about that yeah I'll take a passing the pilots refers to them yeah so from our perspective the the unnatural base pair itself appears not to be generally recognized by damage repair pathways and MMR brn er um what what we think and so none of this is published yet but we've now looked at a whole bunch of different sequence context and some of them are better replicated than others and it turns out that for the worst replicated sequences SOS plays a role and if you make a Lex a uninduced will strengths that all these go up so we think and that makes sense because all of the other pathways will involve recognition of the nucleus and presumably they just can't read so they're probably undergoing fuel cycles are probably maybe recognizing a distorted duplex or something and but they can't doing about SOS first different because it's damaged specific independent damage type into them um the more subtle question about damage proximal where the same thing is that happens by and you prevent repair in approximately um sure but you gotta remember all of those strains are viable at least for reasonably long periods of time because that sort of mutation they just don't accrue enough certainly on the time scale of a protein expression or even frankly a long term evolution as firmly as we run them we just wouldn't expect that to get to know all the corners of dixon is going to to annex near to the plasmid system x rays is producing enzymes is producing fix the enzymes also which is in fact a completely isolated system there i don't think there should be pushed you'll have a very short question also about don't have orthogonality thing abs are you sure that the DNA and RNA they have two spiral structures but if you change the background you can get these river like things what are you do you think this really kind we've worked into a feasible information clarion system as presumably reasons for a DNA to have this this particular spiral form but the blood is indeed by the polymerase it should be completely that of course the helical structure is shown and it's more it's also more folded when I stay for mobility so it's hardly again or the system which you will be able to use in chromosome completely replace you oil in it okay it should be restricted to something where it is public / you cannot go as far as I I think you cannot
do much fun as a plasma that's also reason yoga perhaps to comment on this it might be that yes double helix is wombley manteca biology so but when you look at the choreography of DNA innocence you see that there is an awful lot of times energy expenditure and so on to unfold it so it might be that black bones that are prone not to make races but that would you know induced fit like a fly toddlers be able to replicate nevertheless might lead to write because that would be much much more easy to to to manage and evolve at least XD so it might not be an inconvenience after all not to a proper folding in organizers say that makes me changing the backbone you have a whole generation of possible student ID that yes it might be that dumb prank I'm sorry yet left you have you have a whole choice of possible structures this silo is just is just as acquisition example what you have to see what has to be processed what has to be done but what is it task of this change to change topologically change documentary yes again it's against the question and if you use it in certain for certain functions I think it should be used in the social certain functions what look can you be more specific I would like to know what it's a different functionality but what do you think of what is your freedom but the dream is is 25 again is to do to encode in this in this artificial nucleic acids for reactions from vision the whole cell becomes dependent to put headquarters in the cell to control the solar functions to catalyze some solar functions in that system and that of all sellers defendant who capillary totally new functions you put in this artifact Atlantis fracture pattern and it's not balanced it's about optimality if you if you have a helical stew if you have something with a helical structure like DNA you produced in itself it will recognize DNA RNA and block the nation functions as a prop it's like there are several reasons for that you could think of non-party lien biology like so you something like that you know try to implement systems but i'm not occur spontaneously and that are not in the axioms of molecular biologists just for the sake of it it is scientifically interesting to do not there is another answer to your question sir which is that when you say what are our other natural specifications respected by this unusual stuff and so on frankly the the notion that we have of natural design racial classification is very short so making artificial systems and interrogating natural systems with such systems as Floyd acceleration is a way of understanding so synthetic biology is not only a way of constructing top of table one is also matter a way of understanding life as we knew well and I grabbed it more than his eyes because something has to be optimized it is it's a better shape or better structure then I understand me you oughta modality chemically organizing might be a prophet this is among my fellow Englishmen relatives that's not a discussion right now about when illusion optimizing something and efforts if it does what is it of it one sec York American so it's not always so obvious thank you it's to a question about I think nothing is not funny that you can create these women like DNA structure my friends with them the question to me is well why has nature not already experimented with this before my name is this place but then you already claimed that he somehow has try to do that but he didn't take it because it wasn't successful now you know if you argue develop a question is there any plan to to try to make to put if he rose and you're basically the same organism I think it should be up to Mt I think not I think the way I think of it and a chicken home comment we've actually talked about this um this it i guess i don't i don't think they would ours and the trose we work together because i think that dumb I mean we've looked at lots of Miss pairing and we occasionally in the process we actually in evaluating candidate robe a Spurs we've course evaluated what in the end would amounted to miss pairs between our analog and other hydrophobics water excludes all water really well it doesn't oil oil at schools water oil doesn't exclude oil all that well Rico has it sorted to your pages or recognized by his place so we have a vote based better systems together I don't think they would be orthogonal I think that yeah I said so I think that they would probably go on at some intermediate level against each other maybe they're each commercially available so we could each of us could have bought the other guys so obviously there's had a question we've heard from George George trying to pack as much information as possible in in DNA the user does alike at a long-term storage of computerized information and then the unnatural base is that you're incorporating I guess announced ability certainly against biological degradation they also enhanced ability against physical chemical methylation in other words would these be better formats to store information like for eternity I think that would be better because as well as well the systems which means for this museum with its modified days are splitting and an artificial backbone you don't have it right cause the default anymore they are totally much more stable on DNA so DNA is one of the most unstable only the new pro ties you can have once you change the under stranger basis because he cannot have any protonation any more than that I in base because they have no not eaten animal can go so they are intrinsically kind of the most people asian a backbone side and look like as much much much more stable so your answer is yes if you want to store information better do it with with excel with modified basis and the worst you could take its DNA but isn't isn't a question of that DNA needs to be because even if a most excellent DNA need to be mutated and changed and also so isn't the fact that the instability of DNA is inherently part of the evolutionary process well I think it's significant I'm just laying it out there but that's that's why you have value half your services instead of timing basis in or me because people claimed it already become a deal it only makes and uracil makes more mistakes and I'll start dinner so there is much more evolution of power normally and dinner which is place which is based dependent area of course displays again well i think that people argued about this for a long time and I think the one argument is that it just was um it exceeded sort of a cost ratio benefit to optimize to the point that it optimized because they probably could have optimized further right um but I think like the ability evolution the ability to evolve the evolutionary evolvability is a pretty cantankerous subject and the
evolutionary biologist gonna wind up about and so I bet but i think that dumb as a result it certainly drives evolution and it's hard to imagine how it could happen without is the perfect my channel is joseph emerged in my life and i listened to all the stops about to judge anything with genetic code is my colleagues for doing this earth evolution they have all these machines you can put the animals and they made you cut the code so you could you get this machine can you put it up but this bed about anything it's reached a code for you and that's its produces the genetic code well actually it turns off but it's become very clear is in many cases this code it's not sufficient and not all of the information of individuals use to create a phenotype six encoded in DNA it sort of the field for epigenetic is now a big thing and so I was one electron extended has been known whether this alternative basis are also prone to things like methanation and that sort of thing what extent that kind of can be modified this way but then in natural systems there's a whole certified enzyme system that is if you see if you some small modifications put a simple halogen and epitome appearing on pyrimidine or you remove the nitrogen and critic or bottom they are not any mark of lies by restriction enzymes at all I know very small modifications so I think it's going to burn meta dates and very significant so I would give you four are from our perspective sort of two reasons that I don't suspect that is coming on around the corner and what is um they're inherently less functional because they have less reactive functionality in them you can look at the natural appearance and primitives and you see reactive centers um and secondly you know evolution works by tinkering or exaptation or what everyone call you really have to have something that's close to start from and there really shouldn't be much in a cell that recognizes the analog so they're pretty far off so the idea that you know that a small number of mutation or the lateral gene transfer could you know sweep in some new activity it's probably i would guess is unlikely if you made record you know if it's great I think there's plenty for us to do before we really thought that well so we shouldn't forget that there's a lot of epigenetic information if you saw in the relations between proteins are they make for switch or whatever there's a lot of information this is still available what would not be available is the type of information by analogy that is contained in the fact that the science is methylated the disease only that's it I think it's actually more likely that our analogues themselves would act as sort of epigenetic switches by altering histone packing or unwinding during transcription or lots of things like that if you go to artificial system I think you should always go to the most simple system and its its purport an affair to complete it'll completely avoid system and expecting that you would need the same functionalities floor flowing for violence and vinyl yeah so if the goal is to add my god fishel amino acids in your protein sequence for an octagonal system could you both comment on the alternative approach with the vocoder base pairs from Jason sequences how that kind of is complementary or competition yeah yeah so I know Jason well um you know there's to me I'm a interest I mean it's an interesting approach because there's no um I mean ribosome production is tightly coupled to sell growth um it's really hard to imagine a robust healthy cell expressing on our top completely orthogonal set of ribosomes that have you know altered ribosome binding sites to recognize the forward base barcode nonetheless is certainly a creative idea people have been working with wrote codons for a while and of course you get suppression effects as a three codon as you expect um in this case we're not RF media that they're just the natural anticodon mediated um insert so one I mean some people often talk about you know combining this with sort of Schultz and then maybe a resynthesizer gnome that's been that's had more drastic reappropriation done but that's we look at that is you know really elegant and really hard approaches to getting at most to a natural base pairs in um and then sorry I mean oxygen um and that all might work and its really interesting but we think that perfectly interestings or different approaches is to rewire it up from the bottom and have to not worry about suppression as a three base pair codon if you're trying to read it in the four code um or to have more than amber um so there's all sorts of issues about trying I mean people George church try to synthesize essentials and I mean there's really interesting growth effects there because it turns out that essentials are enriched their GC biased writing their enriched in the beginning of jeans and they affect structure and you can't delete them so the that our perspective is the code and codon usage as part of the code is is is extremely as pleiotropic lee entangled with lots of other aspects of the self and it's just i would argue an interesting different approach to try to rewire it up from the bottom where you avoid those clear traffic conflicts regarding the quadruplex coding and so on there is also on it has to be recognized that what has been proven so far is translation of quadruplet between triples you know i don't think there is a demonstration that several quadruplets can be correctly translated another frame the frame has Minard and as flowing said re-engineer endure the ribosomes as to make a multiplet cutting and so on whoever a lot of work that is that is really daunting but I was wondering you know if you could if you could have like 10 different amino acids in a protein 10 10 new ones I was just wondering about to give a hint of the kind of applications we might want either what could we do with then new amino acids that we couldn't do like two or three right now like real application so um 10 new unnatural amino acids as opposed to two or three unnatural new well so first of all the options we have right now is one yeah and there and it's not efficient there's an immense amount of optimization required and remember the whole notion of deleting of trying to repro trying to recode and maybe delete our f1 that will never work outside of bacteria right eukaryotes have one RF that's it there's no there's not you don't have the trick to play there so so the notion and from a from a pharmaceutical perspective the reason all of your natural base pairs are put at the beginning is because if you get truncation it's not as much of a separation problem but if you if you can't do that game over because you can't separate on I mean farmers not going to run columns sure so I would argue this justification for a single now for two there's all sorts of other issues for example in sort of simplest thing if you've got a farm and you ask well what are the two things are interested their interest in a TCS and they're interested antibody drug conjugates and they're interested in well pegylation or parental eicher or right PK issues so right from the start we could give that week the hope is that we could do both um that's a pretty easy argument to make everything else wouldn't start to get but right now people can't do to getting two three you
start to gets up to some more esoteric things like well that plus a metal binding site or something that facilitates purification or um but 10
okay for maybe I put in those three plus seven additional colors by different G of videos just because it would be fun does he call but you might be okay but I think they should be there might be 10 different but but not telling the same time who could have a cassette of two and always two different because the problem with a lot of enzymes is that the number of the reactions they can catalyze is limited so you cannot capillarity is on reactions who can capitalize what off reactions choose read in organic chemistry which is not which is not which you cannot use in biotechnology and they're going to be in the servlet demand for new proteins that can catalyze different reactions for making chemicals for making drugs so I think that is certainly need for more than two but four for generating new catalytic solutions you might only need you to another castle of to another castle of two dependent on the reaction you want to catalyze sort of put the continual in this election I think after an initial we should stop a distressful like you tomorrow one tomorrow morning they are actually are going to continue on this because actually had I had a bit skeptical tour except we actually can design the next step I think if you it would be a big thing if you could make an alternative genetic system but then you would tell you would me to have it evolve at will to weed out all out on the other although other than the mistakes that is it so may excite our that's so this is so this is something which I suspect will come upon you tomorrow also new in one of the top was one question in the back country I just wanted to comment on that question that there's two companies petty dream and a pharmaceutical to choose a whole spectrum of unnatural amino acids in pet by discovery but in an in vitro transcription translation system so if you can carry which arch or enzymatic recharge of trnas with whatever you want you can reassign the codons again whichever way you like and because it's in vitro you don't have to worry about poisoning the rest of the sound so in that case certainly for peptides circular peptides specifically you know d amino acids are obviously advantageous and so on and so on so i think there's no shortage of cool things one could do it here once the cat has been expanded okay i think you as a proposed that we will continue this discussion tomorrow morning and i'd like to thank both speakers this afternoon