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Bikard, Ellis, Boeke: Discussion

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Bikard, Ellis, Boeke: Discussion
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Meiosis biosynthesis inductive platinum gene genetic information spliced Estradiol restriction originators strains chromosome Muraminsäure Combining P sites cell Altbier transcription defects protein Sauerrahm overexpression parents level Aroma compound biochemistry type concentration Maische fluorescence Intron genome Gletscherzunge PolyA Tails ligand Cocaine synthetic conditions function coupling Ionenbeweglichkeit Silencer (DNA) compounds Priel screening yeast
chain fine domain gene phage Ribosomal DNA chromosome clones organizations Centromer genome stability end Receptor systems model repeat resistance sequence amplifier bond potential Atenolol case strains cell defects transcription overexpression active site report tool Färben type Proteomics DNA physiological Chemical oscillation Transposition <Biologie> variability mutation syndrome conditions metabolic pathway Telomerisation chemical structures Filter nuclei screening
type Transposon mechanism tRNA steps translocation gene genome case man systems strains Unterdrückung <Homöopathie> metabolic pathway materials protein defects addresses screening amplifier sequence
chain activities sensitivity mechanism factors current gene methyl electron stress cell control localization modifications Library period type probe Chromatin DNA dynamics assemblies Histone Topic genome systems synthetic Eisfläche conditions metabolic pathway coupling Silencer (DNA) regulation yeast screening addresses form stuff
antigenic DNA recombinant inductive bond Meiosis Transposon activities domain gene case chromosome terminal transcription factors Offener Leserahmen amino acids active site Butcher level Digital elevation model tRNA DNA genome systems Hypobromite synthetic metabolic pathway Transdermales therapeutisches System remove yeast screening sequence
all right questions Devon yeah you mentioned at the beginning that one of the interesting questions you can pro is what you're not slicing is really essential and I was wondering if it's if it's not possible for alternative splicing to still some of some of these silencers etc excellent so in yeast we have only five percent of genes have introns and the fast fast fast mobility those at one in front so they're essentially is no alternative splicing in sarah busy I there there are a couple of genes that have more than one introns and one of those shows alternative polyadenylation but that's something we could easily refactor so I would never recommend trying this with your favorite mammalian or platinum so Jeff oh go ahead while we're waiting up so if I understand you right for your redesign you were able to do like there wasn't really any part except for the one exception that you said that you weren't able to redesign and even when you were assembling all these you Denver had a problem I mean are you saying i guess i'm curious like what was the biggest thing that you couldn't redesign they were surprised by maybe the size the example you gave actually so yeah I usually spend a lot of time on this at the beginning what I talked and I didn't 10 you today but we spent almost nine months thinking about what would be the smart things to change and what was too risky I had to go for so in fact so far nothing has crashed due to the fundamentals of the design um one change of plans that we made after krause all three gross ohms six and chromosome 8 were already designed and underway was this paper came out from a group that had first said you can delete introns with impunity from many genes which is true statement there are some exceptions to it so we leave them in for now there came a second paper that and and we didn't know yet whether sin 3 was going to work the second paper comes out and says actually if you delete ribosomal protein introns and there's quite a few of those there's often a fitness defect associated with it so we actually pivoted and said okay from this point on we're going to leave the ribosomal proteins in the introns in and then come back and take them out later you know very carefully because it turns out you can get around these fitness defects by fiddling with gene expression levels so that I say that that one is probably the one that it's the one place where we actually made a change on the fly yeah but everything else we haven't changed the original design so it's very consistent from chromosome promising yeah so oh yeah almost a social engineering question you were able to get all these different groups and countries to all agree on the same design so is it a uniform design across all the chromosomes yeah that was a mandatory aspect of the project so you have to get your own funding that was that was one second one was we had to do the design so that enforced you know design County and the third one was you had to agree to give away all the screens and all the DNA sequences without restriction basically to any you know academic or industrial partner so those are the three major things and some we got some pushback on some of those but we didn't basically didn't budge on those three principles my questions so you can think are quite love with this individual basically with I'm wondering what happens if you put this strain into competition with the metro trains right so um very good point so there's fitness and there's fitness right so we have a very nice way to do cocultures and measure the fluorescence the rfp and one gene odor and so we have that sa up and running and we've so far applied it to chromosome sin 10 and seeing no defect after many cycles of co-culture now that's under one condition of course and i'm sure we will find we will find conditions where it's not going to be his fitness wild-type but we have also seen some instances of a parent gain-of-function after scrambling so if we one way perhaps to get around fitness defects will be to do some light scrambling and select for things that we cover fits that was one of the things that we worry about and prompted us with Lucas trembling in one box sex with your simple victory no problem as sexy as hell they so far and they've never been selected for meiosis right but when we put them through meiosis so far all of them have been proficient at it we're going to make 50 retro with the wrong time that both both to themselves and to the wild-type yeah I'm not sure we've done that with all the synthetic chromosomes we've completed yet but definitely for chromosome 9 are three and six we've done that experiment if I were to look for an explanation but I only find one type in your motivation it would be somewhere there you can need to conform to the to the network configuration so you can put function up with you you can scramble as you wish but that you want to make the photo time series oh I'm not talking about the scrambled screens I'm just talking about the bass strings you know okay remember the scrambling is something you do after you finish the synthesis so after scrambling there will definitely be strange that have and meiosis defects I have no doubt about that so you shouldn't we use a scrambling probably you get a lot of cells that die right yes and you would expect that this effect would be even more pronounced when you start combining all the difference synthetic chromosomes because you are doing more possibilities to mess up with the cell is that something you're worried about that's a point you'll even a short induction let's kill everything well we have two knobs we can turn one is the short time and the other is the concentration of the ligand the compound that activates the cream so in fact what we do now for our evolution experiments were involving these strings is we actually run them without the estradiol and there's a tiny bit of background scrambling and a little bit of leakiness and and then there's another knob we can turn is the promoter that's driving the creek so I we're pretty confident that we'll be able to handle album but it is definitely a valid concern it's not a question of our jack so as I understand it bio studios as a software to design genomes or both automatic related at an annual way and my question is whether it has been I mean the designs or threaded in design in order to use be all the recipe project is it flexible enough it is in fact tom has been very frustrated because we have he hasn't been able to get it to run on his computer but I'm happy to tell you Tom that we have an AWS instance that I think can be working anywhere so the other half is ascended to anyone it is completely generic is based on gbrowse which is the generic browser or you can do j browse but we mostly you to browse so it should be quite generic and we've even done circular bacterial genome they're just as a demonstration question hear me oh actually sorry I forgot there's been a request to have the question repeated so um I apologized so from now on I we can repeat the question before you answer it Thanks right so soon you Jackie admission 21 fitness conditions that I was wondering for maybe genome or chromosome 3 if you look at kind of transcriptional profiles like essentially profiling profiling things
like that do you see if you know maybe it doesn't matter but I'm just curious whether you see signatures that the places you change there are changes or right so thing is asking did we do transcriptional profiling or other types of profiling and of course we did the vast majority of cases there's essentially no change no statistically significant change to the expression you know other than you know subtelomeric genes quote unquote that we deleted which are no longer there so they come well on volcano clock I was telling you earlier we have done more limited proteomics on some of these strains and again occasionally like the one I showed three four we turn something up but it's probably you know on the less than one percent of the time model yes I had the question about the bacterial crisper um so when you deliver these systems you back to your pages so have you observed any resistance coming up against enough against the crisper bigoted against the back day of the delivery itself yes so you you would definitely expect to see different kind of resistant to this strategy so you could the crisp air every simulation in the target and also blog the entry of the crisper by just mutating the receptors of the other page capsids and we haven't observed that so far and the reason is quite simple is to this event you expect them to happen but at a relatively low frequency and in our case we see survivors at much higher frequency than the interpretation of the survivors will serve our just cells that did not receive the crisper at all and that's just because if you even issue deliver 100 times more phage we particles themselves will always have bacteria that just don't receive it and we also observe that there is actually some some by ability doesn't we expect it to follow some sort of course own distribution but it actually does not and it indicates that probably some cells tend to have more receptors and other self in two populations there may be some variability that also explains why some don't receive it three questions so first the new chromosomes are they real chromosome like linear chromosomes with Tito mayors and ours and central mayors and things when we see them one at a time they're just like a minute dunia chromosomes have arses centromeres and two years and the answer is they always have arses and centromeres and we can make them either in a circular format in which case the only telomeres for we can make them linear we describe a little tool we call the telomere ater which is a little device you can insert into any circular chromosome and then when you cut it reveals to seed sequences and very efficiently establishes linear chromosome the second question was on the ribosomal DNA regions and you said that they insulate the chromosomal regions and I wonder if you could tell us why that could be or how that could be oh right yeah so how the question is how does the ribosomal DNA kind of insulate two domains with a chromosome and I think the nucleolus is a gigantic structure right and so at at one end if you will the DNA is coming in from the centromere and at the other end it's going out to the telomere and those two sites are not close to each other at all they're very far apart in fact so as a result those chains are really separated from each other by I think the nucleolus asked me to be about close to a third of the nuclear volume and so I think that's that's what makes it essentially behave like to separate ourselves from that perspective related to that you showed that other chromosomes you would have potential in certain sites that from the ribosomal DNA or from and what is a potential insertion site any phosphodiester bond you want it doesn't disrupt a central team so it couldn't hear it in theory okay what's nothing special it not the true side pocket no nothing special I had a question as well about the brambles genomes so you see these copy number alterations so am i right seeing that most of those are like direct repeats in the genome and my question was obviously be how about the stability these things point you selectively ever just propagate okay so the first question is are the three peaks of tandem repeats and the answer is they don't have to be in fact many of them are inverted or partially inverted which is a signature of something called a rolling circle and with all in circle amplification which have been described for the two micron circle yeah and we also see what we call transpositions where a copy just jumps in between into another box P side on the same from Salt crazy and your second question was stability of first village three so we haven't really studied that at stability of the scrambled screens very extensively but what I can say is that it's stable enough that we will determine an unambiguous genome sequence from shore tree sequencing okay but that's about all I can say so they've had like 30 generations yeah 30w yeah but of course that doesn't mean somewhere in there you didn't have small clone that you're under wife something we do know that if you pick the little colleagues they are often very phenotypically unstable so there may be some ongoing instability there perhaps from die centrex or something like that so we have decided to leave those alone yes I did not understand what you said about murdering human disease and password transplant these this fine and yet per usual patient disease his auto was a all news what I didn't get right so the question is just to clarify what I said about patient mutations so there are many many case reports in the medical literature correlating a neurological or other disorder with the existence of patient mutation in some gene right because the person found this mutation and they think it might be associated with the disease but they're very descriptive and so we've actually were two for two on this there were two reports of mutations in this pathway look easy they were rare you know very rare complex neurological syndromes in one case and I can't remember the detail of the other one did not you know something that's arisen torture one and and the data you know were limited and what we did was we moved that exact mutation into the east and still we cannot distinguish a change with phenotype okay but that is one explanation don't you think because this happened several times especially I don't remember the details with mouse and it turned out that the physiology that the pathways of the mouse is different so you cannot prove the filter phenomenal commutation of the defect because you your choice the choice of your organism is wrong and then I think the way to to cure this is to humanize
that the whole pathway now and then you find what you right so I totally agree with you first of all but we did humanize this entire pathway human I did every step from you know starting materials to amt every step is catalogued catalyzed by human hands on that being said it doesn't mean that for example the protein doesn't fold exactly the same way or something like that so it's not not by any means to finish but it was surprising to me that zero left two cases if we recapitulate any defect so evolution thought Jeff and I were to jay-z primacy to on misunderstanding something in general but I was reading about aging and it says maybe it's connecting to a solidity so did you look at making your list when could you conquer all of that oh that's going to a party it and productivity right the question is whether we've looked at the aging phenotypes of any of these strains and the answers we haven't but it's a great idea yes could you please make a comment on the choice of the recipient string you used to make this using an instrument okay so question is what is the screen we chose and the answer is a dy 4741 which is a special favor to bunt because we constructed it in our lab but it's also isogenic with the string that was sequenced and so our whole you know we're building on the shoulders of giants okay all the sequencing that was done was done on strains very closely related to this one and we didn't want to take a chance on you know drifting to another sequence and running into problems related to background so that that's really the main reason it's it's a totally horrible screen for doing anything practical you know the industry and so you describe the leg off chromosomal rearrangements and natural isolates of puppies how does that get together with the observation that tRNA genes and transposons are high i recommend right so why did you take them out if you don't have recognized okay so right but I can see why you're asking a question so why did I say I'm the one hand that tRNA genes or hot spots for genome instability but there are hardly any natural translocations and the answer is that there are many studies where people have done evolution in vitro and kima stats and forbid a stats and things like that and when you do that you often have translocations or duplications and a vast percentage of those are due to change it you know they occur at tRNA genes so that's where where the data comes from the natural ones also some some percentage of them also occur to your yes oh sorry well I've got a question for you actually so that you you constructed this flip-flop but this genetic flip-flop system in your and Yuri multitude gay tries to pedophiles upon your an epigenetic mechanism for right when you look at so the interaction networks in fact it appears Omar do you see similar things it was a similar flip-flop type of fun so the question is the flip-flop toggle switch style network which we used in using has been used by others in bacterial systems do you see those in natural network yes they are seen I don't think they're seen in particularly large numbers exactly in that Forry entation where it's two things professing one another other than maybe in lambda phage switch this is a common well-known example if I
remember reading there's a long k singh ji sexually is one case is yeah pseudo highfalutin i will switch ok not the the major gene but the world the two wanted a bistable repression anything i think like like those who knew but he never
spoke okay okay my question Layton so both of you talked about gene
essentiality and deleting genes so you could do that with crisper into a screen and scrambled can do it you can get down to middle of genomes and previous previously with yeast people have done knocking every single gene out and now knocking pairs of jeans out look at the sensuality as well but i think i read recently that there's a sort of it's not a black-and-white system that someone should i think it was in yeast that you could not jeans out and then depending on how long afterwards USA whether the cells have a sales are getting on it can kind of recover so a gene that should have led to an essential t if you let the cells have some time and then the cell can survive without that gene as long as it's got a period to adapt so zone see I can see regarding the I think whispers currents for sure are not going to be very well adapted to to see this kind of a phenotype because you to do things you know no pool you only have a certain dynamic range of depletion you can observe and if something is depleted strongly at some point just disappears library and you cannot see if it covers later so yeah I think probably for this kind of thing you will still have to go manually or maybe with some ice recruits like Jeff is doing all right I heard a talk on this at the east india summer is quite interesting i think i think the scrambling could be a good way to accelerate the discovery of genes that allow bypass of that special class of essential genes that's only essential in the absence of some other generally changed so I am actually very interested in that topic I think a related question that much harder to answer is how many different ways to cells die Jim can I pull it that up because it seems to me that the dynamic aspect of networks which years electrons talking about with either metabolic networks that you can dynamically adjust to different conditions after perturbation and find different you know stable states is a characteristic and we heard talks by that earlier in mammalian cells there's also the issue of of chromatin based and auto assembly or autoregulation for epigenetic control as well which is clearly you know these scrambly express express such as egestas was something like that is going on in the East genome and you've got any plans to implement probes for that kind of behavior I'm thinking for example a dks 9 type print approaches to recruit chromatin modifications or something like that we can create specific modifications and then potentially follow changes they might propagate our time repeating that question but it I think part of your question which which was also asked before that I didn't really address was do do we need to worry about are we recapitulating the epigenetic states that would be found in the native gmail right well let's move the mechanisms approach and how you get it get it those more directly right so I I mean I think the general the question is really important especially as we look towards modifying or come feels where we have DNA methylation and histone methylation of flex attea that you don't having these in the East were very fortunate in a way that is a stripped-down system and teal mayor of silence names and HM local silencing are very well known to recapitulate for naked DNA within the time it takes a calling them grow but obviously there's a lot more you learned on that so we are very interested in introducing other forms of epigenetic regulation from mammals for example into yeast but yeah it refer to be an interesting interesting here to go tom yeah see you know we thought about that when we started first working with Tala factors may be other people have now gone on and show them both with tell effectors and d can sign the recruitment of silencing and these kind of things and that could be part of creating synthetic subset of eric regions with metabolic pathways and control the silencing of I have the whole coordinate regulation in that region of silencing and activation the tell the factors stuff you talked about it looked like it operated over a range of ten fold like there was a high background yeah so is there is that a real background or how are you going to deal with that that's that's a real background but part of it is so we have improved on it in the latest version I think it's more like three people but it generally comes from the fact that with intentionally this promoter system we're using is a week promoter because when we overexpressed how factors because we want to use this system to chain tell effectors in logic systems to get when the overexpressed tell effectors we got a lot of stress on the cells we're trying to work with kind of a weaker system this modified pfy one murder is a lot less than something like our great honor sorry there's been a couple people
waiting dem apologies in the East in many genes that are good virgin overlapping did you met any overlap or you separate the open reading frames so question is how do we handle overlapping genes and it turns out when you look hard the vast majority of overlapping genes involve a so-called verified open reading frames with a so-called dubious open reading frame and so for example we had many instances where there was a TIG codons we had to recode so we applied the rule always change the dubious or in those in those instances once you go beyond those instances they're very very few bona fide overlapping genes there's there was one that was really clear cut with two verified works overlapping and we had a TIG and it couldn't change it without changing the amino acid so what we did was we went to nature and we looked at whether there was anything in nature that had a cheap energy ta a mutation and sure enough there was one so we just wanted that change I had a question getting back to the humanized protect bonds mehbeh pathway I was thinking back to a sin parkas talk where he was talking about can reconstitute NN kind of having the minimize to the point where I was all in vitro so I was thinking in your case for the humanized system was it just coding regions or you know you know in the future if if you were trying to reconstitute it you think you have to be tweaked you know promoters and things to get levels and so on all right so the question is when we did the pathway transplant how did you do it and well that's a slightly took out so we took the human or potent optimized is produced and then we assigned to the Gila north the corresponding yeast promoter and terminator so the regulatory sequences are yeast the coding sequences are human and of course paying that's that's a limitation and you might want to play with that in the future but unfortunately human promoters generally don't just work and you Steve so that would take a lot it big brush gently southern question yeah it's a question is whether can you can you maintain two chromosomes identical chromosomes that might be in different states so hopefully you've got lock sites taking each gene the idea that you might override local control by at ds9 with a repressive domain targeted to each gene on chromosome so you could have active chromosome solid chromosome and flip back and forth between each one by induction which is gets back this idea making substantial burden basis which you then follow the consequences of innovation that's a really cool idea so you're saying target the loxp sites with the d cast line and then essentially add I saw where you have one native chromosome and one sign is your bro sites as well as except curves only three and be returning have this check and see if there's any Pam sites in velocity of sight yeah yeah and be made diploid of of your strengths and do you see any egg here they do recombining meiotic Lee yeah and actually that's that's the problem with trying to combine multiple synthetic chromosomes you get all these patch works out but we come up with a way to homozygotes the synthetic chromosome so that basic comes a non-problem but they definitely written by we're working on building recombination maps to see whether the frequencies of recombination along the chromosome are similar to another question um I remember I I heard sort of scuttlebutt that the blattner screen where he remove Beltran's bosons as soon as you introduced any foreign DNA or basically sort of start contaminating the genome back again with transpose ons a bit cute hey does the same thing happen in yeast and B have you seen it okay I I don't know about the scuttlebutt I hadn't heard that but um in arc so questions when you bring the transposon back in will it yeah or r transpose on topping from your native East chromosomes into your synthetic okay so to that point do the transposon jump from the native to the synthetic that was one of the other reasons we made the party chromosome because as we build the individual chromosomes we take out the trna gene so all the targets are gone because those transposon love to go near trng so that keeps them clean but I'm cosmic but well they will go into the other native chromosomes but they won't go and elsewhere on the synthetic so they always go to the Terran a site well ever say Holmes yeah yeah yeah so the thing the worrisome part is what's going to happen when we you know start reducing the number of native chromosomes because these transposon copy number control systems and they're going to start getting like really antsy and jumping like crazy so but we we have a plan to help with them which is we know all about the transcription factors they love and we're going to destroy one of those two you know incapacitate them when we get to that face that's a fight okay ah any last questions for almost out of time okay perfect Flynn thanks very much to all of our speakers