 Thank you very much. Thank you first. I'd like to thank the organizers for giving me a chance to invite me to this nice Conference and also giving you a chance to talk about some of the work that we're doing in the group So let's see if this works. Yes. Okay. So all of you know that there's a really big problem with antimicrobial resistance There's lots of hospital infection 3 million patients in a year about 50,000 deaths 25% of those are colonisans hospital subsequent infection and Resistance is rising. So here this blue line is MRSA. You can see it started lots of increase Even enter a cocci which have resistant to vancomycin, which is the so-called antibiotic last resort has Emerged and is on the rise and you can see the proportion of MRSAs In some of the countries within Europe that are quite high But this problem is not just for Europe, but it is absolutely worldwide So let's think of antibiotics. So who makes them? Well, they're Actinomyces more than 80% of all commercial antibiotics are produced by Actinomyces Actinomyces are Ground dwelling ground positive soil bacteria. You can see a scanning EM picture here This is the soil and you can see spores coming out of the soil If you take a grow in the lab a colony of Streptomyces or Actinomyces You see some colonies like this growing on an agar plate on the top You see these blobs and these blobs are antibiotics being secreted from the cells here if you cut the colony in half here and Look at it from the side. So here's the agar and you can see inside a pigmented Antibiotic red pigment and antibiotic being produced and in this case, it's being retained in the cell here are some structures of antibiotics or natural products you see penicillin to Vancomycin we just talked about vancomycin just now Daptomycin, this is the last really approved drug and in fact, I think a lot of people you know already This year's Nobel Prize for medicine has gone to three people two of them Campbell and Omra Were involved in identifying and developing evermectin So this is drug has an anti-parasitic activity and used all over the world and the last person here, too was awarded because she Refound artamidicinin so she was going through old literature and in the Chinese literature. She found that people used A sweet wereward a plant to cure against malaria and then so she started extracting Artamidicin and we'll come back to this story in a minute Okay, so the point is that these natural products. They're very diverse in structure Some can be simple and some can be very very complex Now the first antibiotic to be discovered was penicillin in around 1940s So-called the miracle drug in those days and in the 1960s we had a really lots of antibiotics found novel antibiotics This was the so-called golden age of antibiotics, but then after that we've had a steady decline But on the other hand resistance has increased So why do we have this decrease of antibiotic discovery? Well, some people say that the pharmaceutical companies are no longer interested in natural products development Why well it doesn't make a lot of money So if you want to get any drugs on to the market, it costs a lot of money Antibiotics usually you take it for two weeks and you're cured You don't have to take it anymore If you're suffering from cancer or from heart disease or diabetes, of course You have to take it for much longer time So you can see what's why the pharmaceutical companies don't really want to work on antibiotics anymore Some other people say that okay people don't work on these natural products because actually there's nothing to be found out there We know that's not the case because we've genome sequence and actinomyces. It's called streptomyces clavilligius This is in fact a commercial producer. It produces something called beta-lactamase inhibitor So if you take any beta-lactam or amoxicillin or whatever There will be these beta-lactamase inhibitors in there as well So when we sequenced this the first thing we found shockingly enough was that we found 50 in total 50 potential secondary metabolite gene clusters Now clavilligius is known to produce about five We know the structures chemical structures of them some we don't know but we know that they make around five or six So the rest the 45 44 of these gene clusters are either asleep Not being transcribed or translated or they're producing in such small quantities that we just cannot identify them And in fact, it's not atypical for clavilligius because we've done a global microbial genome analysis And we find there's a lot of secondary metabolite gene clusters out there So if you look at this green part here The tall the bar if it's as tall then you see that there's lots of pathways in these organisms So this is actinobacteria here and you can see that there's lots of secondary metabolite gene clusters But in fact in all of the other microbes. Yes indeed. In fact, they do have secondary metabolite gene clusters Now if you look at the blobs these blobs show you how novel these Structures can be and in fact the actinomyces Actinobacteria's of course they have lots of secondary metabolites, but you can see that the blobs aren't as big It means that they're very similar in class. In fact, these other microbes might have much more Diverse chemical structures Okay, so we have done a proof of principle trying to wake in some of these clusters So this is an orphan gene cluster found in a streptomyces species called streptomycecele color This gene cluster was identified right before the genome was sequenced In fact, we found a few enzymes, but in the end because of the genome sequence we could identify the cluster and In fact, we didn't know that there was a secondary metabolite gene cluster Apart from the ones we knew on here And so what we did was to delete this repressor and by doing so the mutant started producing this yellow color the parent usually uses produces blue color and now it's producing this yellow color and in fact We could also show that this gene cluster was responsible for a compound that has antimicrobial activity So this is showing a bioassay that we typically do for looking at antimicrobial activity So you put a lawn of basilis This is ground positive and then if you see a halo like this it means that this streptomyces here This is a patch of streptomyces is producing a compound that's killing off basilis in fact The yellow compound the structure has been elucidated by Greg Joseph's group and you can see that it is absolutely a novel structure So you can imagine going back to this figure You know if we could awaken all of these potential secondary metabolite gene clusters We're surely to have novel activity and surely find novel diverse chemical structures But if to do this how we can how are we going to do this if I have to delete one gene or Activate a promoter or take all these heterologous pathways into a different host It just really doesn't work. It really never works and of course, it's time-consuming So we want something that's more systematic Something we can do high throughput and we can design and of course we want to use synthetic biology So there was a lot of definition about definitions on synthetic biology for the past few days My definition for synthetic biology is that it's going to be the next industrial revolution it's going to be the biotech 2.0 and Basically, it's to engineer new life forms with unrestrained versatility Which means that your imagination is the limit for using synthetic biology Some of the examples I want to also show you because this I think gives you a flavor of synthetic biology You'll be hearing from Jeff later on tomorrow about synthetic genomes. And of course, you know the story about Craig Venter Institute using them like making the genome from microplasma so you can actually synthesize chromosomes But one of the favorite projects that I like to Give as an example is those from the iGEM competition So for those of you who don't know what iGEM needs iGEM stands for International genetically engineered machine competition. So this happens every year. It's done by undergraduate students There's more than 300 teams international teams coming together to compete every Around October time. So the undergraduate students work over the summer period to produce our microbes usually microbes sometimes eukaryotes using standard parts So here's a little bit of an example for example Heidelberg one year produced an E. Coli that could recycle gold from electronic waste Groningen University. They made a bacillus subtilis, which is a Biocensor for rotten food. So, you know, we do have sell-by dates on the foods we buy But that doesn't really correspond to the real use-by date You don't know if it's okay or gone off and of course we waste so much food now What they wanted to do is to really have some indicator by sensor where you can actually really know whether this food is edible or not and they took meat as an example so they engineered the bacillus it turns blue or purple if the meat has gone off and they actually made a prototype So they had spores of bacillus Surrounded by media in two little plastic kind of chassettes And if you wanted to activate it you kind of squish it so that the media touches the bacillus And then you put it on your meat and if you put it on rotten meat it absolutely went purple So this is some of the ideas that they have so I think these iGEM projects give you a really good idea of what One can do with synthetic biology Another example of course all of you know who are synthetic biologists. It's about this Artemisinin So this is work from Jay Keasling's group and I just spoke a little bit about before because this is the Novel prize not Jay, but the lady who found Artemisinin got the Nobel Prize So Artemisinin is made from sweet wormwood and what Jay wanted to do was to produce it in saccharomyces cerevisiae Why because for plants the production rate is not always consistent If the weather is good you get lots of plants if the weather is bad you don't get much plants Okay, and also it's time consuming to get you need lots of plants to be able to get a lot of compound So he decided to take some enzymes using enzymes from yeast itself But also some from plants and to make a precursor of Artemisinin which is called Artemisininic acid and then using semi synthesis to get the end product So this has already been on the market. It's actually free Since I think two years ago now. So people it has been distributed to people who have malaria Another example I want to bring up is vanilla. So everyone knows about vanilla vanilla is a mixture of Tastes and the main component is called vanilla and here's a structure So the real natural extract only takes one percent of the market. It's very very expensive So all of the ice creams and biscuits and whatever you eat that has vanilla flavor Is produced from lignin or coal tar. Okay So a company called Evolva said, okay, why can't we make vanilla in yeast? And that's exactly what they did and they have now produced vanilla in yeast And I think last year they already started selling it They they're aiming for 75 percent of the market. So that would be great if they can get that far Now at this point I wanted to bring this up because I know that paul Talked a little bit about it, but I think it's something as a synthetic biologist I don't know if it's to do with mathematics. You get to this problem as well, but the problem about public perception we had a lot of of Problems when the genetic modified plants came out. We don't really want to do this again But you can quickly see that how the NGOs this isn't friends of the earth I think pick up all these kind of new developments It's something we have to be aware of we should be able to talk with the people engage with the NGOs And try to say look, you know, we're not really doing something bad We're not trying to to harm the environment in fact It's the other way around because instead of chemically synthesizing vanillin We're trying to make it from microbes, which is going to be much better for the environment And not going to be toxic to the environment as well So I think this is something that we really should keep in mind about engaging the public So the two example Artemisamin and vanillin it's great that they did this that you know We can make these compounds now for using microbes in in large scale as well But one thing what my group wants to do is to take this even one step further We want to produce Compounds that nature's not seen before by using synthetic biology and this is in terms of antibiotics And well, how would we do this? Okay, of course We would use the design build tests and learn cycle concept And this concept will be in different levels. So for example, we were talking about parts So parts is enzymes promoters ribosome and blind size terminators Any of these components we would design first using in silico analysis, we can predict them we can do Simulations and only those that we think is going to be the best enzyme the best caloric activity We go off and build them and once you build it, of course You need to test it to see if it actually works and this of course if it works If it doesn't work we'll feed back into the design again And then on the devices level one can model the pathways we can design the pathways the biosynthesis pathways How do we want it? What kind of ribosomal binding sites do we want? How strong do we want it? Again, we can simulate it and then only use the ones that we think are going to be the best build it Physically and then once you build it, of course you need to go off and analyze it And in this case we can do an untargeted metabolomic analysis Which means that we look at the metabolite that the cell is producing as a whole And of course from this we can go back into the design and Rebuild it so that we get the best pathway On the systems level, this is the cell level We can again design and model genomes And then to take those predictions actually build those chassis Deleting enzyme pathways Making more of them so and so forth and then again testing them and in this case we want to scale it up into bioprocessing And in fact natural products is wonderful to use synthetic biology because it's naturally modular We talked about modules modularity before in these The talks but in fact antibiotics is naturally modular and how is it? Well, I'll show you an example here of erythromycin biosynthesis The core gene the core structure. This is the core structure of erythromycin Requires three huge open reading frames. There's could be about 100 kb large Within these open reading frames you have modules within the modules you have domains and these domains are these blobs And in fact the domains have the catalytic activity and what happens is it's very similar to a fatty acid biosynthesis It takes a c3 unit loads it onto here. So you have this structure here The next module loads another c3 and does a bit of enzyme activity That's this and then another and another so you elongate this fatty acid chain And at the end an enzyme tells you to cleave it off Cyclize it and then it has some sugar modifications and you get your erythromycin And you can see from here that there's modularity here and on this level as well And because it's modular now we can kind of cut and paste and mix things up and start thinking How can we change the actual end compound? So exactly how do we want to do this? How do we want to use synthetic biology for antibody production? First of all we go to all the genomes. It doesn't have to be microbial. It can be from eukaryotes Anywhere as long as we can find the genomes we want to identify these secondary metabolite gene clusters We also want to identify enzymes that has special activity. We want to change the enzyme activity We want to change the substrate specificities and then we want to bring all these enzymes together With a promoter, ribosome binding sites, terminators, perhaps even regulatory circuits But at this point what's biggest difference from something like a biosynthesis that's been done before is to actually rewrite the DNA So if up to now we've been reading the DNA using DNA sequencing now we want to rewrite it using genome synthesis Once you rewrite your complete DNA clusters you want to put this into a screening host screen for the product that you'd like to identify And once you've identified that compound we want to put this into a production host Because the production host is completely different screening host because its primary metabolism will be geared The flux will be changed as well so that we can have large scale production of the compound of your choice So when we think about synthetic pathways, what do we exactly need? Okay, we need Libraries of enzyme parts because without the enzymes you really can't make a pathway You need promoter libraries, different strength, different regulatory promoters, ribosome binding sites And then you stick this together But how are you going to make these libraries? Am I going to do it by hand? Of course not So what we've done is to design develop some software to identify secondary metabolite gene clusters This is called anti-smash and we're on the third version already What this software does is you can put the whole genome sequence or any gene clusters and it will identify like here The gene biosynthesis gene clusters in this case. This was a genome sequence that was put into the software It identified 25 biosynthesis gene clusters and then it shows you the open reading frames that you can find and even the domains as well and It even predicts the core structure of this biosynthesis gene cluster. So this is a web based software Here's the Website if you'd like to use it, please do we're always welcome for feedback to hear feedback And if you have some sequences that you don't want to put on the website You can download the software locally to your desktop and use it yourself Another software we've designed is called multi gene blasts. It's very similar to anti-smash But the thing is it's not limited to antibiotic biosynthesis clusters. There are a lot of other gene clusters out there for example membrane-associated Differentiation developmental gene clusters So this is looking using if you want to look for any genes that are clustered and are conserved You can use this software to find it. Okay So using these two softwares what we've done together with the natural products community is to make a genome annotation standard It's an annotation standard. So we can in fact even use it as a database So we've asked all the natural product products community about all these different antibiotic biosynthesis cluster classes of antibiotics All these kind of different questions and we've asked them to put them onto our database And here What's the good thing about it is that if you find a new biosynthesis cluster and it's similar to something You can go into mi big and actually identify the publications that's been there What kind of experiments that's been done? Who's been doing it and all these kind of information Anyone working on natural products if you'd like to join us, please do we're very happy to have you on board Okay So another software we've designed or developed is on the devices level that that means we're going to the pathway level So this is using mass. Oops. Sorry mass spec data That you get for peptide natural products and linking them with a genome sequence so we can actually identify which is the biosynthesis pathway and predicting that biosynthesis pathway We've also done some systems level design and this is using our model metabolite modeling So there was lots of things about modeling Perhaps I can show you how modeling actually Connects with design and the testing of experiments So in this case, what we've done is a constrained based model of all these different actin my seed strains And the question that we wanted to ask was which chassis which strain is going to make a lot of my antibiotic And so here down here you see different classes of natural products antibiotics And the lighter the color the better the host can produce it Okay, so here is streptomyces here So streptomyces are the ones that produce a lot of these natural products. And in fact if you look across Okay, so for some classes is a very good host birth, but for others not very good Now if you go down a little bit further down here You can see that this is a very good host for a lot of different compounds And in fact, these are mycobacterium species not the pathogenic ones, but the natural wild type strains So of course, this is only in silico analysis We need to go back into the lab and test this whether this is true. And that's exactly what we're doing now Another design Software modeling that we've done computational analysis we've done is on regulatory networks. So we have some Small molecules that we think are regulatory compounds for antibiotic production And if we in fact we've been able to show that it's a by stable switch for antibiotic production And we've also used the constrained based metabolite modeling to try and Understand flux So what we did this was use streptomyces clavillageris the genome sequence that I talked to you about before We had transcriptome data for the wild type and the high producer of beta-lactamase inhibitor So we put these two transcriptomes data together with the metabolite modeling and asked Which pathways do we need for the high producer or we don't need for the high producer? And in fact, all the green pathways the metabolite pathways are those that are redundant in a high producer Which means that we can minimize the metabolite pathway redirects the flux and at the same time minimize the genome as well Okay, now that we've designed our parts. We've got software that we can do use to make our Enzymes and pathways we have to think okay, how are we going to build them? The first question I had was can we actually make enzyme libraries and can I swap enzymes around? And to understand this we used The biosynthesis cluster that produces the calcium dependent antibiotic. This is a non ribosomal peptide antibiotic you can see all the amino acids are linked together and he uses one Amnesty's called l hydrophenyl glycine and in fact because it's not being produced by the organism It needs the three biosynthesis enzymes embedded into the biosynthesis cluster to produce this compound So we took a look at this enzyme here and asked the question. Can we swap it around? Can we is there orthologs? Is there homologs? In fact, there are homologs and orthologs unrelated from arctomyces To test whether these enzymes can actually replace the original enzyme We deleted the enzyme original HMO from the producer strain and then complemented these six genes or enzymes And you can see over here in this panel. We tested to see if they have bioactivity Again, this is a lawn of basilis and the halo means that it's killing the basilis. So yes, it has antimicrobial activity It's producing something but to make absolutely sure that it's calcium-dependent antibiotic We did a LCMS and show that it was indeed the compound that we're after So this tells me and gives me a good idea that yes, we can do this now It's really we can make library of enzymes to make antibiotics The next thing to think about if I have the enzyme parts now I want to put them together Okay, so what's the order that I should put them together remember? We're completely rewriting this. We're coming out from scratch So we decided to use a test case for these six Enzymes which produces this compound here This is the natural orientation of the genes and the promoters that's found in streptomyces But if you think about it because you're going to rewrite it it doesn't have to be like this It can be like this with each promoter in front of each enzyme It can be coupled with only three. It can be in all sorts directions So if you start thinking about this, you have thousands of combinations that one has to test to see whether which one which pathway which orientation which combination is going to be the best That's a lot of work And I don't I didn't want my phd student to spend all his time making lots of these constructs So what we did was to go back to nature nature's been using evolution to get the best out of Producing compounds. So can we learn something from nature? so these are Five different biosynthesis clusters which make a very similar compound to this What we found out was there was two enzymes here two genes that are always always next to each other And in fact, these are two proteins that have protein protein interaction and you can't split them And so now we can use this rule to start off with and say, okay These two enzymes always have to be together and then carry on and start making manipulations And doing this we can try and we're learning What is the best way of constructing a pathway? What's the best promoter strengths? What are the rules that we need to follow to actually design these pathways? We're also doing this refactoring building pathways using another compound, which is monotropines So as you saw from the previous slides antibiotics are a very complex structure So we wanted to use something that's a little bit more simple And for that we decided to use something called monotropines. So monotropines in this case, this is limonene. Sorry, it's Covered up a bit now. So monotropines are used for flavor and fragrances like mint Flavors lots of this smells lemon smells grapefruit. All of these are Very close to monotropines. So what we've done now is to and monotropines by the way is made from plants Not from microbes. So what we had to do is to get enzymes from the plants And also using some from microbes as well to produce this limonene. So all these enzymes here What Adrian's done is to put these pathways together and of course we had the challenge just like jaded How do we put them together? What's the best way of putting them together? What's the promote? How about the promoter strengths? Does it have to be always very strong or does it not have to be strong? So Adrian made two different constructs like this and decided and started testing them Of course, we tested for translation Here as well and tested for different strengths of promoters And then also induction levels because these promoters have can be induced by different compounds He looked at induction levels and to cut the long story short what we find is strong is not always good It looks as though the promoters that are a bit weaker is much better than having very very strong promoters In fact, this is still on a plasmid and this is an E. Coli by the way And the plasmid is is quite a high copy number plasmid What we're doing now is actually putting this onto the genome to make it much more stable And also it seems that it works much better this way Okay, so we've written the genes we've made the pathways And is that all it is for synthetic biology? In fact, it isn't there's other things we can do other things We can engineer some of the things we can do in terms of spatial control We can make synthetic protein scaffold or compartments making compartments We had a speaker in the first day talking about compartments We can also make microglucon sorcia So if we want to use synthetic biology for industrial biotech Really make it cheap. We have to have it cheap the end product has to come down in price And the process has to come down in price A lot of the things that people are thinking about now is not using glucose as carbon source but rather lignin Or other waste products and for that we can use some microbes Which are very good in degrading these things converting into glucose and giving it to somebody else who can do, for example, biofuel production So these are the ideas for microbial consortia So one of the things that that we're doing in my group is making compartments And you've heard already nicely about the compartments. I don't have to it arise now But just the idea that if we want to express a pathway It's much more nicer to happen in a compartment because the intermediates don't get degraded If you have toxicity you can overcome that So what we're using is a Bacterial micro compartment BMC from Ute EUT So what this compartment does Naturally is to degrade ethylone amine But what we want to do is get rid of this pathway here and just make this core Empty shell and we can do this by expressing these five enzymes and into this Empty shell what we want to do is to express monoturipines Now monoturipines are volatile compounds and this is all done work done in E. Coli And if you try to express lots or produce lots of monoturipines E. Coli just dies It just cannot cope anymore. So the idea is to put this biosynthesis cluster into the BMC shell Encapsulated so the E. Coli is not so toxic and it can grow much better So ash has already made these constructs. In fact, we have some preliminary Evidence to see that we can see These BMCs being made in E. Coli Another thing we're trying to do is what you need is a tag to encapsulate them These enzymes into the empty shell and we've been able to synthesize some of these target Sequences and ash is also testing these as well Okay, another level of things that we can do in synthetic biology is to control its expression So it can be a very fast Control like an allosteric control or just just in time. So you only have the genes expressed when you want them Or you can have signaling molecules. We can synchronize the cell growth So in our group, we're working on small molecules called gamma-butyl lactones And these are found in actinomyces or stratomyces, in fact And if you look at the structure, you can see that it's very similar to acyl homo-serial lactones So AHLs have been used as regulatory circuits very well in E. Coli And what we'd like to do is to use this as an alternative to AHL or complementary to AHL regulatory circuits So Marcus is starting to use the circuit into E. Coli and see how far we can Produce a nice regulatory circuit to be used Okay, so now we've designed everything we've built our Pathways we built our chassis So now we're going to produce everything fine in huge amounts But normally it doesn't work like that and if you're engineering a chair or a shelf It's the same even with computers or cars Sometimes you need to tweak it And so our favorite way of tweaking is metabolomics And we're using the untargeted metabolite analysis using the high precision lcms And to to show that metabolomics really actually does work. We've done a proof of concept Experiment and this is Actually inducing an antisense glutamine synthetase So what it does is when you induce it it stops growth. So you make a synthetic switch And this is the wild type. This is the switch and at all these time points we actually Got all the metabolites and look to see how the metabolite profiles change I think you were saying that you can only do seven experiments. Well, we did lots So you can see here we did five Six biological replicates. That's the growth curves within the six biological replicates We had five time points each And then we had two lcs Two different lcs and we had the positive and negative mode as well and we did three technical replicates So that's quite a lot of samples that we tested But by doing this we could actually see a trend though. It gives you a variability in lots of From each of these Samples you see variability, but if you do lots enough of them You can in fact see a trend and you can see some of the compounds like this one here Is immediately reacting to the synth induction. So is this one? Well, other compounds amnasis here Are only changing when there is a stop in growth So these are all the metabolites that we saw that changed. Okay by Proterving this this is the antisense glutamins in the days So we don't really understand why this happens But one thing we can say is that all metabolomics is a great debugging tool because it shows us the cell What's happening in the cell as a whole? Okay, so What do we need for synthetic biology and robotics? production We need parts by synthesis genes from different sources We need to engineer the chassis Circuits controlled of gene expression not just on transcription but translational levels We need lots of computational software and modeling and analysis that feeds back into the building And we need to have these analytical Roots And of course this Shows you the design and build test concept. We really need to do this over and over again. And in fact This is not just for antibiotics. It can be used for any high value chemicals or functional metabolites And at this point this was my group my intention of doing it as a group So there's one phd student or a postdoc work on different things But to get it to the next level we need to do this high throughput in a much larger scale And to do this we're doing this in our synthetic biology research center So we've been awarded from the bbsrc and epsrc the research center, which we call symbiocam, which is on fine and specialty chemicals and of course we're Taking that design build test cycle concept and it's housed in this building here called mib in manchester So what do we want to do? We want to access wide range of chemical diversity Rapid delivery and of course we want it to be predictable And we're using this design build test cycle and platforms In fact, we can do this in Manchester because we have a lot of expertise So we have lots of these all these rpi's professors who are involved in our center There are people like Doug Kell, John Loop and Pedram Mendis who are systems biology experts We have Nigel and Nick Turner who's our biocatalysis experts We have also Jason who's an expert in antibiotic production Let's see. We also have Roy and Perdita who's Absolutely experts in mass spec and GCs So because we have all these expertise in house This is the reason why we can actually get this center up and running And so what we've done with the center is to get the money that we received We've made a platform all the money has gone for equipment These are the all the new equipment analysis equipment that we have Also making getting lots of robotics and design softwares And of course to run all these softwares we need people and here we have now 12 SEOs appointed Neil is upstairs as well He's got a poster on a little bit more about the design of this what we're doing in the design platform Please have a look at the poster. It's on the over here So we have also people in place and So now our ambition now is to take this into really a higher level making into a production level so that we can even have real Collaboration with the industry and to have a product in the end from synthetic biology And last but not least I like to acknowledge all the people involved people in the group symbiocam team as well Nigel's group All these people here are Infimeticians I have a very good Collaboration with the infimeticians and I have a project with Companies as well looking for novel antibiotics and here also on the monotherapy project And I'd also like to thank all the funding bodies and thank you for listening Thank you very much. I think there's time for two technical questions The microbes from biology you still have your golden your teeth Sorry Gold in my teeth. Yes, because you said the microbes taking out the gold from the Right, right, right. Yes Ah, yes My one question just to fine-tune what you get can you couple it with selection mechanism of bacteria? I couple the selection you can couple it with selection of bacteria to Fine-tune what you get yeah, because you get to the roughly and then you want to fine-tune For example to against given pathogen you want to make antibiotics and to make circuit With a selection it's possible to make it to make a circuit circuit. Yeah, of course you have electronics analysis And then there is selection from bacteria and then they go together. Yes It's possible to make a cupping. Yes, I think so circuit as in circuit in the microbe, right? That's what you're thinking. You know, I say, okay, you should have your sauce off the way you prepare this Exactly and then see what happens. Yeah, yeah. Yeah. Yeah, so the idea is really designing something building it And then testing and then coming back and doing it. So we had some discussions in the previous sections Yes, you you you need to have a good selection You need to know what you want if you don't know that then you can't do this You don't know exactly the shape of a chemical, but you know what pathogen exactly exactly. That's right That's right, that's right About the bio compartment or the mini compartment strategy So you want to produce the toxic end product? And this is supposed to accumulate in those compartments because in a way it's just postponing the death. Yes, but then Even postponing it you get more cell mass and then you can produce more other compartments that you could actually kick out of the cell um Not not these BMCs, but then on another way you can Kind of purify them a bit easier as well too. You can try and extract them But the most important thing is actually just even this delaying helps with the cell mass It's just getting much more biomass because if you don't have biomass you don't get production. So that's the idea Uh-huh, yes So can we make it you mean? No, it was made before penicillin the first was not okay. Okay. Okay. That's what you mean Okay, okay. Okay. Okay. Yeah. Well identified. Let's say identified not produced but identified They were in similar to the fight a long time ago before you know in in in 19th century. It was a name Flaming gave the name to penicillin, right? But therapeutic use came in in 39 by grimicillin and 41 from mincerin Penicillin was only given a name just as I was known there for for ages. Uh-huh. Okay. Yes. Thank you It's tricky history. Yeah. Yeah, okay. For therapeutic use It was first Grimicillin was a couple of years before Penicillin. Okay, right. Okay. Thank you I have actually a question for questions If you if you want to produce antibiotics or look for new antibiotics Usually you find a lot of them which inhibit inhibit the growth of other organisms procurates But most of them cannot be used because they have negative side effects on humans Is there something in your procedure which Tries to to take this into account that you do not develop hundreds of potential candidates without No chance. It's all to do with the screening method Of course, you can put in lots of different screening methods I just put anti-microbial because it's the easiest but of course you can put in Toxicity screening and all this in there as well too. So the nice thing about this is flexible Whatever you want to do you can plug into it There is no universal way to fight toxicity. It's all to be experimentally Toxic, there is no chemical way to say whether it's toxic or not No, of course not. That's why you need to do screening like using mammalian cells or whatever. Yeah And then we will hook up very different for different, only it's been selling toxic for example for Yes, that's right. So some industries want broad spectrum, some want very narrow spectrum So it depends on what you use as a screening Okay, thanks again