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Technologies for engineering the microbiome

Video in TIB AV-Portal: Technologies for engineering the microbiome

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Technologies for engineering the microbiome
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2015
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Understanding the role of the gut microbiome in modulating host health and disease will require technologies for localized and long-term monitoring of microbiome and gut functions in vivo. Furthermore, new strategies are needed for precise modulation of microbiomes to enable new diagnostics and therapeutics, since existing approaches for modulating the microbiome can have significant off-target effects. Synthetic biology can provide new tools for studying and manipulating complex microbial communities. We have created strategies for engineering commensal gut bacteria, such as Bacteroides thetaiotaomicron, a major and stable member of the human gut microbiome with synthetic gene circuits and we demonstrated that the y are still functional in mice stably colonized with the engineered bacterium. This work provides a resource for Bacteroides genetic engineering towards future applications as non-invasive diagnostics and therapeutics in the gut microbiome. Furthermore, we have created technologies for the specific knockdown of bacteria living in mixed microbial communities. For example, we engineered CRISPR-Cas antimicrobials that kill bacteria based on their genetic signatures. In addition, we have built a technology platform for engineering phage host range, which enables the creation of well-defined phage cocktails that can kill specific subpopulations of bacteria within mixed microbial consortia. We anticipate that these strategies will be useful for the targeted knockdown of bacteria in complex microbiomes to understand the functional role of these bacteria or achieve therapeutic effects.
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set Shine-Dalgarno-Sequenz constitutional bind gene Gletscherzunge end man P sites function Sekundärionen-Massenspektrometrie RNA color consensus coli active site Library translation sequence
Ancient Roman units of measurement phase bear factors Bohr secret nitrogen man specific Strength protein transcription overexpression active site cells coli level Darmstadtium Bock genome phosphate Sekundärionen-Massenspektrometrie Gamma RNA Peroxyacetylnitrat Förde coli consensus basic sequence
inductive sugar Sulfate sensitivity Bohr capacities Plasmide bind operation Memorial gene Stöße Recombinasen Chromate Klinisches Experiment Strength Rhamnose P sites color Actin Library coli clones derivatives basic milk sugars concentration gels gene genome source complexes Memorial end systems coli regulation Lactose-Operon sequence sense Gum arabic activities man Zearalenone Combining transcription control overexpression active site cells Inneren level translation type Entsafter DNA carbon Gletscherzunge variability Dermatansulfat induced chromosome Combining Rhamnose function overexpression Luciferasen
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Probiotikum Cross bind phage man stress fiber gap tool cells coli Capsule (pharmacy) C-terminus type phage los thickness end Combining synthetic van Tee orders of magnitude Gamma coli cells clones chemical structures
van Graukäse Bohr operation phage source firm man
thank you very much to the organizers for giving you an opportunity to speak about what we do in the laboratory of Timothy Lou I apologize he cannot be here so he sent me instead of him the lulav is a large operation with many different aspects ranging from biomaterials to the UK lyrics in bio with activities in cancer detection and treatment and also production platform and is also involved in bio computation so building all of those gates thats in bio is so interested in but what I'm going to talk about is the last aspect that the lab develops which involves a possible applications of Earth's in bio in infectious diseases treatment and microbiome engineering all of that being somehow linked together the human body is not just you chaotic sales it's also a very very large amount of microbes trillions of commensal microbes and a very diverse type of microbes the gut microbiome for example is completely different from the skin microbiome which is itself completely different even from the head microbiome and what I'm going to focus on today is the gut microbiome and the microbiome especially the gut microbiome is heavily involved in a lot of different aspects of health that ran from development to immunity eliciting at the very beginning of life digestion mood behavior and so many other things that are still to be discovered but unfortunately the tools to manipulate it when some problem is discovered I extremely limited they are basically limited to wiping it out with antibiotics possibly introducing pro or probiotics although their efficiency is heavily debated as they are and once you've wiped it out with aunty it may be replacing it with the healthy microbiome of someone else and that's about all we have as of today if you want to replace a micro body so transplant change the microbiome with something completely different which may
or may not be healthy what we want to do is to have a much more delicate approach to microbiome engineering we'd like to be able to either selectively add a bergh with given with predetermined functionalities to an existing microbiome or possibly remove a Berg which is involved in a pathogenic situation and this requires tools that do not exist as of now so the ID that we
want is there may be a problem somewhere in the get you design a bug which will then be ingested travel through the stomach all the way through the intestine reach the site of inflammation cancer you name it and then elicit a medical action to cure the problem as I have written here going from the mouth to somewhere in the gut requires passing through a lot of different environments and this is where most of the previous applications of microbiome engineering probiotics and so on failed very few bugs are capable of adapting quickly enough and well enough to all those diverse environments to actually still be alive when they reach the site where they may do something so that's one of the challenge what we expect that's
Cinderella biology with its capability to design desired functions inside of microorganisms can help with designing microbes that will face this this is a very common description of synthetic biology which is usually considered to emanate from a concept which leads to a design the design may be modeled and simulated and there will be none of that today and then it is constructed probed in vitro in some form of isolated culture measured possibly evolved if something is not optimal so rare right and then if it's your fails maybe go back to the design go to you through another model iteratively until you manage to build whatever it is that you really want it to be and at MIT we an engineering school so we are very much for that top bottom approach where you have an ID and no matter what you will build it lots
and lots of work on somehow modifying the microbiome was based on e coli the vast majority of the probiotics that exist today I either call out or lactic acid bacteria Cola is slightly better than lactic acid bacteria but none of them really colonize to get microbiome easily you bet sorry gram-negative of the type of e.coli or maybe something in the range of 0 1 percent of the gut microbiome so it's almost negligible it's not completely useless but very much so so we are looking for other
possible bacteria that would be more suited to creating a probiotic that would be stably maintained in the gap and one search bacterium is a bacterial it is theta iota microm and you will forgive me if I just call it theta it's way too long so theta is just like coli or gram-negative bacterium it's an obligate anaerobes but it tolerates oxygen pretty well it just does not grow bit at least it does not die it's a member of a dominant filer in the microbiome it accounts for about thirty to forty percent of the total number of species in an average human gut microbiome it is very abundant up to ten to the ten cfu per gram of stool in again an average human microbiome and is present in the inner large so in a large amount of micro biomes of humans and finally it is relatively amenable to genetic modification it's not nearly as easy as coli but it's feasible the problem with B theta iota micro micron is that although it is amenable to genetic modification they are hardly any tools yet and suddenly nothing like we can have in coli where we have banks of various inducible or consider you've promoters large amounts of plasmids which are compatible so that you can add gazillions of circuits inside of the exact same cell so the question is how can we transfer all of those different biological computations so those are examples of biological computations that we devised in the lab so the lab devised various methods for integrating memory inside of bacteria we also devised ways to make analog computation we worked also in digital computing like many others we also devised ways of rewriting DNA directly in vivo inside of the cells and all of that was developed for coli but how can we transfer all of that into theta when we don't even have a single promoter which is properly described so the first step in doing what we wanted to do was to develop a set of basic promoters it usable and and consider leave a set of ribosome binding sites the very basic set of tools that you need to build any secret into any living organism we started from that simple plasmid developed in the garden lab P&B YouTube which is an integrative plasmid in theta iota micron that integrates here catalyzes its integration into a sarin ti na the good thing about that integration inside of assets aren TI is that they only two in theta so if you can activate one with your construct the other one has to be here so that limits the possibility of having multiple integrations and it is selected with air through meissen and it's a suicide plasmid obviously in thia and it has he Nana look which is a smaller version of luciferase that gives you a readout for anything you clone in front of it so what we'll do from that P&B you to sequence is to very ribosome binding site sequence just in front of nano look very promoter sequence as a luciferase activity in different conditions and see what we can do and finally engineer all of that to see if we can change levels
in collide the ribosome binding side is well defined it has a consensus sequence of a GGA ji ji that matches well to the end of the 16s RNA all of that is good and if you modify the sequences on both sides of the ribosome binding site or within the ribosome binding site you can change the level of expression of genes from 0 to maximum which is whatever color is capable of tolerating in
bacteria this the consensus sequence of the ribosome binding site is not that different it matches to the end of the 16s RNA chest as well and so the idea was well if we do exactly the same thing as in coli then that should work right that looks feasible and even the natural color ribosome binding site should work well enough so there shouldn't be too many problems with translation so that's
exactly what we did we took our pnb youtube plasmid it had we cloned known constitutive promoter from theta in front of it the shine-dalgarno was that of that BTW 1311 gene and we started making libraries by randomizing the sequences around the shine-dalgarno and assessing function so just for reference this is the kind
of thing you can get in coli when you modify the sequence of the ribosome binding site so you get from very high to absolutely zero with some of the mutants you make interestingly enough when you do exactly the same thing in theta you go from high so that red line here is the natural ribosome binding site the one we started from so we can increase a little bit maybe half a log we can decrease some we can decrease by about two log but there is no way to make it completely negative for some reason you can put any sequence and no matter what you will still get some level of expression some people suggest that actually the pairing with the 16s RNA is not the major determinant of specificity of the ribosome binding site and that in theatre it's more binding to the s1 protein of the ribosome which dictates recognition so there's a lot less of the base bearing involved as recognition of an actual phosphate backbone rible phosphate backbone because I don't think anyone knows what it really bites nonetheless we could still get something in the range of about three logs difference between the strongest and the weakest of our ribosome binding site but it this large difference in behavior between coli and theta probably explains why a lot of parts that were brought from color into theta completely failed because they are obviously immense differences in how the two bacteria work now we decided to look
at promoters in coli promoter specificity is dictated by a sigma factor there are several in e.coli I'm not going to go into the detail each of those Sigma factor recognized consensus sequences which are different and so exactly the same way as you could do with the ribosome binding sites earlier if you modify either those consensus sequences or the sequences in the middle you can vary the strength of the parameters theta has a completely different promoter architecture there is a single Sigma factor which is responsible for all transcription in the in the genome of the bug and the consensus sequence is pretty different from that of coli with instead of a minus 10 minus seven bucks and instead of a minus 35 or minus 33 box which does not look very much like that of course so when you have a secret that works in coli it's pretty unlikely to work in theater directly because if the ribosome binding sites may possibly work there's no way transcription will ever work so we started building libraries of
constitutive promoters using the randomization strategy that I told you so this is our starting promoter pbt 1311 we also clone a few other promoters from B theta that we're expected to work well and we also started introducing variability in all those various regions highlighted in blue here either replacing those regions with those from other promoters or from completely random sequences and I'm just showing you here a few examples of the promoters we got out and all together the different considering promoters we managed to build span about two logs of magnitude in expression level so when you combine the different transcription levels were capable of reaching with the different translation strengths that we capable of doing with you the ribosome binding site library we get about five log difference which is about four logs more than whatever was possible before but constitutive expression is still fairly limiting a lot of the constructs we use for any secret require being capable of turning things on and off on demand so we wondered how we could obtain inducible systems bacteria bacteria is theta iota micron theta has an extremely varied array of carbon source usability it is capable of degrading a lot more carbon sources than coli and just like coli it senses them and induces the genes for their degradation only on demand when the carbon source is there so there is a large amount of systems that can be borrowed from the genome for sensing at least carbon sources one such example is the rhamnose regulation system in theta rhamnose sensing functions very much like our aveeno sensing in kalai so you have a single regulator cauldre are that binds to ram nose and then activates the rhamnose operon promoter PBT 3763 and so if we clone that promoter in front of Nanda look you can see that while we increase rhamnose concentration we increase expression of nano Luke of luciferase and we get an about 104 fold difference between absence of inducer any juicer which is pretty good we screen the genome for other types of inducible promoters a lot of those carbon sources are sensed through kinase response regulator systems two component systems although those of theta are different from those of coli and one such example is the chondroitin sulfate response regulator BTW 33 34 which sensors chondroitin sulfate activates the promoter and we got about 60 fold with that system or the arab in ogle actin system encoded by PBT zero to sixty eight which gives us about 29 fold induction between absence or presence now we had a number of simple parts we want you to check to what extent we could build more complicated parts so try to import functions that are not native to theta and the most simple system to do that is probably the lac system of color so we wanted to see if we could like I to function in theta so we put like I and the control of theta promoter pbt 1311 actually and then created a mutant of the pbt 3111 promoter with lack operator sites at various locations and we build actually all possible combinations of one two or three operators interestingly they all worked they did not work great there is limited a dynamic range in those promoters but what's interesting is that by modifying the relative position of those lac operators we could change the sensitivity of those promoters so that gave us the possibility to induce at various levels of iptg finally we tested whether all of those promoters that have different end users but remember they're all sugars or derivatives of sugars we're orthogonal to each other that is if the inducer of when system would induce another promoter and what you can see on that graph is that there is induction when you combine the the inducer of a given system with its corresponding promoter but absolutely not any induction with the other inducers so they're perfectly orthogonal so now we have a good library of parts for transcriptional control and we
wanted to see if we could go a little further in complexity and what we've done is to see if we could replicate a memory integration inside of theta the idea behind that memory integration is pretty simple it uses the capacity of integrators to flip pieces of DNA between inverted recognition sites for that given integrators and we used a system developed by our neighbors in the void lab where they build an array of different integrates recognition sites along with all of the integrators that specifically recognize you of those site we only cloned four of them as a test trial and checked what happen if we would transform theta carrying this on its genome with plasmids constitutively expressing the the integrase and what these gels tell you is that in the presence of the corresponding integrase you get the right the expected inversion so everything works great we then checked briefly if it worked with an inducible integrase and to make a long story short it does it does actually beautifully much better than in kauai because we have absolutely no activity of the recombinase and when we don't add any inducer which is rare of collide promoters the vast majority of coli promoters have some leakiness especially in those systems still pushing further
in complexity we wanted to check if we could benefit from all of the advances of crisper Cass so I'm expecting everyone is familiar with crisper Cass but in case not very briefly Cass nine the natural Cassadine is a nuclease which requires a small RNA to direct itself to the appropriate sequence in the DNA that it then cleaves here we're using a decays 9 which is a deactivated cast nine so the active site of the nuclease were modified so that it can no longer cut DNA but it can still land on it and stay there so what we were hoping with the decays 9 based system was that we could perturb expression from genes the first system we built who is pretty simple it used an iptg inducible d cadd9 we would clone whatever guide RNA we need to direct a decays 92 given sequences within the promoter or the coding sequence of nano luke and then we would measure the sifrits activity as a function of iptg and what this graph or this graph they both show the same thing show you is that it works beautifully when you add iptg then luciferase activity goes down indicating that the decays 9 really does land here and does prevent transcription it's a little surprising though that even the single guide RNA is targeting the coding sequence of luciferase still had an activity again this is a big difference with coli in coli decays 9 really only works if you target within the promoter and as soon as you get outside of the promoted quickly see is working we then wanted to check again with that system if we could target and dojin streams these two genes here are involved in polymyxin b resistance so we wanted to check if by using the decays 9 targeted system we could inactivate those jeans and thus decreased resistance to polymyxin and that's indeed what happens we also use that exact same system the targeting some of the carbon source usability operands of of theta and you can see here that when we induce the decays line system with iptg then growth on fructose stops pretty abruptly indicating that the targeting works as expected everything tends to work pretty
easily in the lab in culture medium very often when you go to a more natural environment things stop working so we wanted to see to what extent all of those parts that we had built would still work with in some form of natural environment and we chose lies because they're easy to manipulate so what we did was to treat mice with stratum icing to strip them off of their of most of the microbiome then remove antibiotics colonize with various theta theta strains that would not have synthetic constructs and then treat those microbiomes within users as needed collect the stools and then measure luminescence and measure the presence and abundance of theta by qpcr the first system we tried was a very simple arabin ogle actor and induced Lucifer a system just to see if anything will happen whatsoever and what you can see here is that this represents times when there is Arab you know gal actin in the water of demise and you can see that there is a sharp increase in luciferase activity in the stool as we in use and it goes back to you baseline very quickly after we remove inducer so it seems to be to work well we then tested the crisper system in a similar system so it's exactly the same system as I as I presented earlier it's just a lot more complicated so there are a lot more places where things could go wrong nonetheless it still works ok we're probably reaching the border of what no longer works but here again we have iptg edition and you can see that when we add iptg luciferase activity drops which is exactly what we would expect when it stays perfectly flat in the system that doesn't have all of the regulatory systems so here I have described an
additive system to add a program the microbe inside of the microbiome the problem is doing it still requires a pretty heavy treatment with antibiotics to strip off everything that is not really desired and replace it with what we want so we are now focusing our efforts on trying to build systems to selectively remove a given member of the microbiome and replace it with what we a subtractive approach to a microbiome engineering our first attempt at that was using again gasps nine this time the real cast nine the one that cat's DNA and was aimed at targeting NT micro antimicrobial resistance gene in natural environment so the you is pretty simple you have delivery vehicle in this case HM 13 because it's very easy to engineer that m13 delivers a piece of DNA that has the cast nine the tracer iron RNA which is required for cast iron functionality and most importantly arrays of guide RNAs that direct cast nine to their target and the target may be in the genome or in plasmids and in most cases in bacteria when you make a double strand cut the cell just dies it's particularly true on plasmids because most plasmids have toxin antitoxin systems and if you make a double strand cut very quickly the antitoxin degrades and the toxin kicks in killing the cell so results can be seen here here you have all of the control strains that means they do not have the target for the Cassadine system and you see that there is absolutely no toxicity so if the target for the guy died and is not here nothing happens however you can see here for example that in a strain that has ndm-1 the phage that targets the ndm-1 sequence kills the sales here you have a similar system targeting another type of intimate of anti microbial resistance genes and exactly the same thing happens it is very specific to the sequence and you can even mix the phages together and target both at the same time if you want you can also direct the Cassadine cleavage to the chromosome this is what we've done here we've isolated a mutant of our test trainee mg 2 that is nalidixic acid resistance so has a mutation ja rahe this is a single base pair mutation and the guide as you can see is extremely specific it kills the mutant it does not kill the wild-type what we've done next was to see if we could counter select various factors so here we've been targeting the intamin gene that we've heard about yesterday i believe which is a major virens factor of antero haemorrhagic coli and once again with the proper with the proper guide RNAs women age to eradicate the targeted cells with our engineered phages and we've also looked at whether or not these engineered n13 phages could help with survival in the case of infection we've used a wax worm model here and we're following death of the worms after being injected with either a non-sentient sensitive cell so bacteria that cannot be targeted by our engineered m13 or cells that can be targeted with the intamin targeting page and what you can see is that if you don't infect the wax worms don't die if you infect them with just the phage or with sorry with a phage which does not target the strain that we are also putting in the worms they die if you treat them with sm buffer so just any buffer they die but if you treat them with the intimate targeting page which is the one that can kill off the bacteria that we injecting they survive significantly better there is one major flaw with that project and 13 is wonderful for molecular biology it's small it's easy to to engineer you can do a lot of things with it the problem is it's absolutely f dependent it's a conjugative plasmid and f is extremely rare in nature so this is a very nice system in the lab absolutely useless in real life so we have to find other delivery vehicles the system can work but we need other delivery vehicles phages are probably one of the most efficient system for delivering DNA to bacterial conjugation requires way to in made contacts to be useful again in the context of a microbiome where you have billions and billions of other bacteria so the probability of encounter close enough between your donor bacteria and your recipient bacteria is way too low so classically if you want more features you isolate them from nature and you end up with a wide variety of phages that may or may not be strictly lytic that may or may not integrate in the genome that may or may not possess various factors and you assemble them into a cocktail of phages that collectively target all of the possible strains that you need to target the problem with that is that for every fade you add to your cocktail you need a whole lot of tests to make sure that everything works the way you want it to work so the approach that we are trying to use is what if we could transform phages into some form of antibody where we keep everything that makes it a phage that makes it a good delivery vehicle and change what makes it target a given type of strange so that you can develop a set of phages originating from the exact same chassis the targeting different bacteria may be a graphical visualization will make it clear let's say that these are all the strains you need to target to treat any given disease they all have a different ever up they may have different sets of genes inside of them that may provide defenses against phages traditionally you go into a sewer plant or whatever natural environment suits you gather a few liters of the of the effluent and pretty easily you find phage against pretty much any bacteria so that's what you see here you find phages and they may in fact a single bacterium or in fact several and it's pretty unpredictable then if you want to use them in any kind of industrial setting they need to be tested one by one for specificity for stability in terms of storage for their biology they have various factors are they lysogenic are strictly lytic then if it is for a medical application you also need to test safety efficacy delivery all of that one by one before you finally think about putting them all together and have a product which obviously because bacteria where they are they evolve so that cocktail will need to be updated probably twice yearly so twice a year you need to go through all of that for your new cocktail that just cannot work so what if instead of that we use a single chassis a single phage which we have decided is a good starting point make all of those testings so that we know everything about it everything we can and for some of the phages we already have this is almost there and then simply grab from all of those natural features or all of the DNA banks that we have the sequences that we need to make that single chassis in fact all of the strains hopefully then limiting the amount of testing you need to make to get it approved for any kind of medical application obviously at this point this is just a project but actually the FDA at least is pretty interested in it and we have some fair amount of hope that this can be a viable solution to phage therapy in Western world's so let's test it the phage we
decided to use for initial taste testing of that ID is the family of t7 the reason why we decided to use t7 like wages because they are very widespread in nature they're very easy to use in the lab they are well studied and understood they have a relatively short Gino genome with few activities or not too much too many chances for surprises and most importantly they are extremely host independent outside of the receptor the surface of the bacterium the only other gene they need from the host is sale reduction which is an adjuvant to their own RNA polymerase to make it more processive so there is hope that it can actually work in a wide variety of bugs
the other reason is bioinformatics if you align all of the tail fiber genes which is the major host determinant in t7 from all of the sequence t7 like pages together what you find is that the first about 150 amino acids are pretty conserved with the rest is not conserved at all the consensus to explain that is that that 150 amino acid n terminus is involved in binding the tail fiber to the phage who's the rest is different because it infects different bacteria so it needs to recognize different receptors so one of the idea is can we start from t7 or t3 and graft inside of either one of them any of those other tail fibers thus changing host range
summarized here so you take phage a that infects bacterium a HP that infects bacterium be what happens if you graphed the tail fiber gene of gene bit of HB inside of bacteriophage a does it go after back to your be back here you're a or something completely different to start simply we started with t7 and t3 they are both Khalif ages they are both well studied they are both understood but they don't both recognize the exact same receptor well t7 binds somewhat deeply in the LPS of coli t three bites at the very top so that in principle and it's been demonstrated in some cases exchanging the gene should change the phenotype and we can indeed see that here while sorry while T 3 and T 7 infects bistro perfectly well as as well as regular k12 clothing strains t3 here does not infect mg1655 or BW 25 1130 st seven dollars so we now have a scream if we change the GP 17 jeans too I'm going to skip because
time is running out so now how are we going to do that because engineering phages is much more complicated than it seems traditional leaves done with our in replacement systems to make it short because I'm running out of time this rarely works it's long and painful so what we've developed is a system that uses the gap repair system of saccharomyces cerevisiae to completely reconstruct the genomes inside of yeast before putting them back to life inside of colon so the system is pretty simple you isolate the genome of whatever phage you want to play with you need a yeast artificial chromosome which will make the final construct replicate inside of yeast you transform either the genome plus the Yak or various PCR products that span the entire length of the chromosome of the phage with ads from homologous to the yeast artificial chromosome and used by some miracle reassembles that into a fully assembled genome you extract that he start official chromosome transform it into bacteria and if you've done your design properly you get functional phages which you can then maintain grow play with obviously in some cases it's not going to work but at least compared to early replacement methods we have the yeast clone we can at least go back sequence regions check if it didn't work because something happened during the PCR there is a mutation and the phage is dead or is that simply are designed which is wrong and we need to go back to the drawing board interestingly enough that works with a lot of different pages so we've tested it with a number of different t7 life ages and again long story short it works with all of them independently of what hoes they normally in fact
and we built from t 787 that has the t3t fiber and t7 that has just the c-terminal part of the teeth retail fiber remember that only the n-terminus is conserved we had no idea if the teeth retail fiber would be able to bind the t7 cancer by itself so here is again all
of those phages plated on on bl21 which is a common host for them and then
tested against oops a selective host you
can see that while t7 still grows on mg1655 the reconstructed t7 has exactly the same phenotype but 37 with the t3 a fiber no longer grows on mg1655 and conversely for the t3 mutants so the ID works if you change the tail fiber jeans you can change host range we then want you to see if we could cross species barrier here we resorted to a completely synthetic approach to making the genomes it turns out that that phage are that infects your senior only has three point mutation difference in the with the t3 gene 17 so the tail fiber genes so instead of sourcing out gene are amplifying the whole GP 17 we just decided to order a piece of DNA that corresponding to that region where they are the mutations and then go through the exact same assembly process to make a t3 phage with the r c terminus of the tail fiber and you can see here that while t3 does not grow on ya senior t3r grows perfectly fine anya senior and kills it extremely efficiently in vitro
we wanted to go even further and try something a little more difficult we had tried so k 11 here so it's nowhere que llevan is a klebsiella phage klebsiella is very different from Klein that they are all capsulated sharp contrast with the laboratory coli which all have a rather rough phenotype so a capsule is something that a normal day fiber cannot bind you it's a thick layer of mucus surrounding the cell and that prevents the phages that recognize LPS from actually finding it so we wanted to see if we could graft onto t7 something that would allow it to go through that capsule and in fact klebsiella so our first attempt was to simply replace the tail fibers of t7 with those from que llevan but that did not work we don't know why but it did not work so we started trying out all of the possible combinations and it turns out that if we replace the whole tail the tail being composed of GP 11 12 and 17 then we get a phage that infects klebsiella just as
well type so this is t7 on coli Caleb t7 on klebsiella you see does not in fact there are no plaques and here we have k 11 wild-type it does not in fact coli but in fact let's sleep klebsiella and if you bring the tail of que llevan into t7 then you get a phage that grows on clip cell and conversely if you bring the tea 17 inside of k 11 you get a phage that grows on coli so that tells us that although they fiber swapping is a method that can work it's not always a sufficient and that points out to the extreme lack of knowledge we have in the structure function relationship in phages because there is no reason why just cloning the c-terminus of k 11gb 17 would not work there is no bioinformatics way to predict that still it did not work so we are here hoping for as Patrick was saying maybe more predictive tools maybe more bioinformatics maybe in stress that the tools that we have are not sufficient what I want to point out and that will be the end and I will almost be on time is that we used those engine phages in synthetic bacterial consortia to check if they could work just as well as well type phages in removing their specific bacteria in the context of some simple microbiome this all in vitro we are doing animal experiments right now but they're not at all ready so the idea is pretty simple you mix in approximately equal amount probiotic colonies all the targets train for k 11 the targets trained for our yes senior and then you try the various phage combinations and see what happens after a short while and what you can see is that even in 30 minutes when you add k 11 on to that synthetic consoler klebsiella is completely annihilated so on a pie chart like that it's difficult to see but we haven't about five order of magnitude decrease so there is a very tiny cross on here it's just not visible and the same thing is true if you use the the engineered phage t7 k 11 HP 11 12 17 although just as we could see earlier it's not quite as efficient it's probably good enough but it's not quite as good but if you give it a little more time it still works and if you mix t7 which targets yes senior and the phage target in klebsiella then you can you can completely remove the the to pathogens in about an hour that being
said i would like to a no ledge members of the lab as i said the lou lab is a big operation lots of people so particularly here okie ando who worked with me on all of the phage engineering work Rob satori who worked on the m13 Cass delivery fahim and Mark who worked on the bacteria this aspect of things along with our collaborators from the Queen's forward lab at all of the other members of the lab and our funding sources thank you very much
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