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Jung and Isaacs: Discussion

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Jung and Isaacs: Discussion
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so you mentioned that you improve the efficiency of the sense of days with me
Jeff how close you get to the efficiency the wild-type enzyme so it's a good question we're still a ways off so I didn't have the slide that showed the the biochemistry data but um going into the experiment we were about eight hundred fold lowerin activity and now we're about 75 to 80 fold lower so we improved it by by boredom actives will still have more than order packed you to go so we're getting there but we're not we're not there yet and just to give some context we're working basically with synthase synthetases live drive from archaea i'm putting in the bacteria so i think there could be a number of explanations as to why we're still not not only to be but it's still a significant improvement that allowed us to drive many incorporations and turn it into proteins to detail but did you fill this one active citations only or where the full print work so right now it was focused just on like the minimize somebody pockets and these specific domains or the synthesis that will recognize and bind to the anticodon loop of the tRNA so what we're looking at now is yet expanding between on those as well as going deeper into those regions as well because I think both of those two strategies thank you pursued so being able to go beyond so we in those experiments we target about 12 residues I mean in the amino acid binding pocket but if you do the math there's no way we can sample that genetic diversity so we're cuddling that also with competition protein design as a way to better for me in Genesis as well and i'm going to other domains across the protein may I ask you to be the more general question not not technical about this asian i could be struck of what you're safe maybe i didn't understand you said that you increase the resistance to virus pc t7 violence that is one factor because i was an infection and there was a company most a lot of moments and then we were speaking about over saturday you you checked the seven-day period and so you have long-term stability what do you mean by long-term stability you have not the hind type of nature of billions of years and millions and billions somebody spoke about trillions of microorganism so for me that's not scientific absolutely no okay so you're touching on i would say excuse me just be done right that's fine you're focusing on two points really one is genetic isolation right in terms of the virus experiments so what I try to do is motivate fields experiments both biologically as well as thinking about how they can solve real-world problems so that genzyme example was a real world problem and it's a problem that is actually common to all the crowd of fermentation processes right we're about fifteen percent of butter fermentation processes are compromised by phage infection okay so that's a real world motivation to create organisms that are going to be resistant to viruses and so what we showed is data that were 47 infectivity was reduced and some data that didn't show where that achieve expands to other viruses so that what it does is it shows two things it addresses that that problem i described but it also shows the notion which is scientific that if you have organisms then of alternate codes they are not incompatible with genomes of that exist in nature and those genomes can come from other species or the income from viruses which have been evolved over billions of years and what we're showing is that by creating organisms with alternate codes you can create barriers to the facile transfer of genetic material okay so that is scientific the second is on bio containment again that goes after a different motivated by BIOS a complementary problem which is now thinking about engineering GMOs that go beyond your body fermentation processes so there's a lot of people I think we'll probably hear a little bit about this later this afternoon about thinking about engineering microorganisms as new types of probiotics to treat disease for example or for bioremediation so we think about applying these organisms in open system applications one what you want to do is endow safeguards into these organisms one of those actually is genetic isolation another is restricting these organisms such that they can't grow in the wild or in other words you limit their growth to define synthetic environments and what we've shown is that you can basically by virtue of recoding link the viability of these recorded organisms to synthetic amino acids which don't exist in nature coach what you said they are compatible so there can be exchanged and then secondly the containment I think if I understood correctly it's time to the minus 20 but if you have trillions of trillions of trillions and said now a general question about it's not looks / that's not person but my deceased there yeah so i'm not i would like you to hear sure your arms sure so with regard to those experiments we push the limit of what we could really achieve in the lab so if you're growing up trillion cells that's quite a bit of cells and then being able to test those on plates and liquid culture is a big experiment so that's the limit of what we could do in our lab to go beyond that and address
the point that you just raised well as you go to trillions and trillions them ten to the fifteenth cells we haven't tested that yet sorry I can't really speak to that but it's something that I agree needs to be done the quote Kiki unto time thanks freezing time Trevor we
must read it thank you right but we did test it over time full of time yeah we basically tested these over the course of three weeks that's a long time for a graduate student at driving experiments look mention you know are you come back when you have some billionaire later these para nature has resources one can be very stomach you're absolutely right and I would say this only work in two centuries or ski okay I would say this is an important first step okay and there's more than can be done that's okay let's get okay Danielle so I guess building all night can you do a back-of-the-envelope calculation on how long it would take to revert or overcome changes or even adapt by introducing new changes into the genome I mean even while you're doing your experiment here introducing other mutations that you're not intending right that have nothing to do with your experiment that is just arising you know what greater those hearings maybe we don't know what right they need to appear at but at least we can compare what rate those are incorporated yeah it's a good question um I would actually say in the course of those experiments as we were sort of arriving at you know our solutions to the w presented we did observe I didn't have time to discuss them but we did observe secondary mutations that led to escape and we characterize those and actually that was informative in then creating next-generation solutions to actually overcome those problems and so we
observe what you would typically observe you know we observed the formation of amber suppressors we observe mutations in proteins that are important in fidelity the ribosome for example and those were in earlier screams that didn't have the mutations that were targeted at concered functional residues all those essential proteins that's what sort of led to that solution is really going after and approached that distributed the multiple multiple TGS across conserved functional residues so that you wouldn't allow one or two or three who knows how many mutations that Tom that sort of degree of containment going beyond that I think will require larger populations possibly over a much longer period of time that we were able to do I'm not sure Jeff if you have a comment on that as well as I they did some similar experiments but right so the other point to make is regard to the trillions and trillions is that there are many different ways to contain microorganisms so ference got a factor of 10 to the 12th which is probably the world's record at this point i would say well we showed in it in a paper last year that thick you if you use to completely orthogonal containment systems each one contains to a level of one in a million that when you combine them you get about kind of the minus well as you would expect for two independent control systems so there's no reason in principle y 2 3 4 5 orthogonal control systems can be combined that's a good point we actually observed the same results in an analogous system and engineering I can also make a comment on that so a spin-off company of our institute called acta genex thing they were the first last Avenger clinical trials really buddy contained lactococcus for probiotics and so the only thing they were required to do from the regulatory authorities to be able to enter clinical trials was to have an ox 0 traffic classical photography from uracil built into the organism such that the organism had to take a yourself from the gut components in that in those patients listen is the bacteria released and out of the patient consideration of us have dropped so so I to such a low level that they consider this sufficient this was ma approved in this clinical trial I me doing the proof of clinical trial is not the same as a google for real use in the end so I'm sure that we have to build in orthogonal systems to bring that up further but it's not impossible to get these kind of products yeah I think that's really turning what was that a naturally occurring probiotic or an engineered provide a solution for building this inside human psyche on what one do you think are the chances that you could actually manage to building this artificial and they pay amino acid and some vital function for the bacteria itself so then you would make it really depend upon yeah um so we are looking at that into different areas one is to do more molecular evolution of these active sites in these proteins and as well as see if we can further expand and enhance the activity of enzymes by the presence office confe synthetic emil acid and touch them out a little bit at the end and so seeing if we actually enhance their in some way function or maybe things body proteins and your lessons perfectly there's a good bit done you're gonna occur and they don't produce it themselves yep maybe i should add i mean currently we do know almost nothing about how these synthetic amino acids are taking up yeah provide over some huge of excess of these compounds and hopes that somehow is it a leader but curly there's not much known about uptake and I mean availability I mean the costs are immense and I talked to our chemists sometimes it's really difficult to produce these compounds and then at the end we get long Libre so they are limitations to connect one has there been any consideration to refactoring recoding organelles we have a much smaller constraining system basically a translational transcription machinery very few genes mitochondria yeah we yeah we have consider that we haven't done it yet but she would have to worry about changing piece of music they don't think possibly yeah just like that here I was wondering if you could comment on the growth rate of these dRose I know you showed sort of the relative growth rates between our f1 plus vs. our f1 minus can coming up absolute growth rates so the cost is doing the experiment we inherited some secretary mutations that reduce the growth rate to about sixty to seventy percent and the original one that we published that we can tribute to these secondary mutations and as you mentioned we didn't see any impairment due to the art walk out and so what we've actually been doing since since that observation and when that paper came out is going back and actually using an age to revert some of those certain invitations to improve birthday and we have observed improvement and there is it still like I'm us background which I presume would also affect growth rate but I don't know for sure um so be us naka does not affect birth or what it does is enhanced the background mutation bank and so we and others in fact as opposed to out there they're out point everyone to yeah that has shown that you can basically create these dominant negative proteins where you can basically transiently silence new desk as a way to make relax the genome drive high-efficiency mage mutation but then remove those dominant-negative proteins and then stabilize the genome to reduce that back on vacation right so us and others have actually improved that aspect of the mage process yes Victor good yeah what happens to you recorded organisms where they are released into anak 12v darling bottom and like soil or some kind of mesopores or something like that because you know you may have surprises you think that it is completely a certainty of containment and maybe at the end they wouldn't be like I don't know that they need each other so it took a question um so we haven't done that exact experiment the closest we've done that we did this in our paper where we showed the containment is rather than taking a dump of the soil outside what we did is we bought plates that were made from oil extracts and blood extracts and we showed that they weren't able to grow on those so that they were basically able to scavenge from the environment and you know escape through a conventional type of metabolic crosscutting but we haven't yet tried to compete them in nature and I think they were performers were believed to be honest with you same lines I was what I was speaking about was much more dimension with us communications we might not need a clear technologies but along those lines for did you get if you were thinking about industrial strings or unknown domesticated string is there any limitations to your technology that or it's able to be used in industrial strength it's a good question so um as you can see I showed early on the technology is really based on me engineering the replication fork and because that's basically conserved
across biology in principle you can do that and so we have efforts of basically expand this into some other bacteria as well as an east and those are shown to be promising and I also know of another paper coming out in pnas and you should go look at the poster outside that actually shows a system that group from Hungary developed to basically port the mage certain components of the mage process into some of these undomesticated species so you'll actually see some papers come out on this only three months you mentioned that they by removing two copies of the tyrosine tRNA out of three you avoided the emergence of one species of suppressor trna what's the impact on fitness of reducing the copy number of retire so trna five factor 3 and is there some way to compensate for that in this trophy longer right so me were fearful of that experiment before we did it and we only saw about a about a ten-percent drop and fitness and doing those experiments um and i do think that if it was more dramatic than that we would have basically put it under a stronger promoter to compensate for the loss of copy number but for those experiments it wasn't striking enough that wanted those experiments any questions for more sort of a more hypothetical questions in place for using your mother you should be able to swap colors for to nola anywhere i said stupid so have you any ideas about section or listen to do with the very nice I'm not sure I understand your party but you could you share your method see you has introduced a new work an artificial I mean I said that you like if you repeat this two times you collect we swap to do one yeah that's a really that's a really good idea it's something that we have thought about and at least mapped out from a design perspective and I actually think that would create an even greater degree of isolation if you will than what we've done I think it's possible it hasn't been something that we've initiated yet but it's certainly an intriguing idea to pursue and I think I definitely like that better pick up any species the criminals things like sex and yeah it's a way to really fundamentally change the meaning of a code on within that species such that not only would you be preventing the the introduction of foreign DNA but you would also genetically isolate the DNA from this organism going out as well yes question you replace the you a gene for the new 88 what happens if you change the group 8 a-2 um so you're right we converted you a GTO a and that was driven by two main reasons one is the frequency of this proton strategy know as well as how those codons are decoded a translation and so I showed this during my talk but I wouldn't try to confer could convert UAA UAG for two reasons one the number of those protons is on the order of two and a half to three thousand two more importantly is that you a is decoded by both r 1 and r 2 so then the wrapped to not just recoding but then eliminating that function from the cell would be very challenging because you would have to probably knock out the release factor and then engineer the other one to alter its specificity away from you a a so that wouldn't be a natural choice one person could that implementation to change the system because you have to be careful about this you have to check but I mean that's for me the most reasonable thing clearly I've also would do that but you have to take into account s bizarre connection to the phosphate network I mean we just now over the completely different dog he didn't expect this but of course when you think about it for us are and makes a lot of sense fan your comment and question I mean in a lot of ways I sort of anticipated that question with certain things I covered my top where if we do want to redesign biological systems I'll just quote what I have up there we have to do it in the context of evolution and leverage what biology does it does well and is distinct from other sort of more like this thing from other lights a conventional engineering systems and in many ways i think that the exercise in recoding was one way to sort of test a sort of permutation of the evolved natural genetic code so i think in many cases a lot of work that we're doing addresses your overall question but i would say that yeah no i agree i'm just curious like okay so given what you've learned from these experiments like what does that mean like what can we draw more general lessons out of it i would say that really i think the i think the most elegant solutions are going to let's say my come through the lens of engineer but are going to be inspired by how biology solves the problem and I'm a big believer in that so I know that there was some discussion yesterday afternoon on the approaches of modeling versus more evolution I think they both have a roll at the table because we are talking about they offer soul obey the laws of physics and chemistry so there are opportunities to to model as as a component of design but biology always has some exceptions to the rules i think need to appreciate and that's where their ability to adapt and evolve find new solutions or landscapes needs to be anticipated well can they even be anticipated like to your point to kirsten's point about not realizing the links between yeah we might be able to predict what the seasons will be but anticipate that they are there okay okay fair enough a regular session to cut in there I think the design for evolution is you have to consider something that hasn't been mentioned here with this very few labs working on it which is engineering more than one microbe or more than one another species and let them color Roy right i mean that's basically what we see in real evolution is competition with other species and then you have consortium forming biofilms in anything and i think is it's not my area of expertise but i think there's a general observation that if you have mixed populations also implemented this tends to stabilize the fermentation processes in those clearly samples where that is done already so you would have to engineer more than one organism to actually come up with a missionary stable solution that will be my my intuition and then there is a whole body of theoretical biology about competition and thickness in in those replicator systems that have more than one participant so there would probably be a lot of interesting theory to try to ping on the table as well okay well thank you very much for an interesting discussion and with that uh I think we can go to lunch