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Engineering synthetic adhesins and injectisomes in Escherichia coli K-12 to target mammalian cells for biomedical applications

Video in TIB AV-Portal: Engineering synthetic adhesins and injectisomes in Escherichia coli K-12 to target mammalian cells for biomedical applications

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Engineering synthetic adhesins and injectisomes in Escherichia coli K-12 to target mammalian cells for biomedical applications
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CC Attribution 3.0 Unported:
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2015
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One of the aims of synthetic biology is the design of microorganisms with novel capabilities that could be applied for the development of new vaccines, diagnostic sensors, and therapeutic interventions for major diseases such as cancer. This presentation will report the development of two important tools that enable us to precisely program E. coli K-12 bacteria to: 1) adhere to specific target cells; and 2) assemble filamentous injectisomes from type III secretion systems (T3SS) that act as "molecular syringes" for translocation of specific proteins into mammalian cells. Firstly, we designed synthetic adhesins based on the display of VHH domains on the bacterial cell surface with an outer membrane β-domain derived from intimin of enterohemorrhagic E. coli (EHEC). We generated synthetic adhesins against different antigen targets expressed on the surface of mammaliam cells and have demonstrated the specific adhesion of the engineered E. coli bacteria to the target mammalian cells using in vitro and in vivo models. Using mouse xenotransplants of human tumor cell lines expressing a target antigen, we demostrated that engineered E. coli bacteria colonize these tumors more efficiently at lower bacterial doses (Piñero-Lambea et al., 2015). Secondly, in order to express functional injectisomes in a non- pathogenic commensal E. coli strain (K-12), we reformatted the operons encoding the structural proteins and chaperones needed for the assembly of filamentous injectisomes from enteropathogenic E. coli (EPEC). Our synthetic operon constructs lack secreted effectors and regulatory elements found in EPEC. Five synthetic operons (sLEE1, sLEE2, sLEE3, sLEE4 and escD) were integrated into different sites of the chromosome of E. coli K-12 under the control of an inducible promoter (Ptac) using a marker-less strategy. We demonstrated that the resulting strain, named Synthetic Injector E. coli (SIEC), assembles functional injectisomes upon induction with IPTG able to translocate proteins into HeLa cells (Ruano-Gallego et al., 2015). Collectively, these results open the possibility to target specific mammalian cells with engineered E. coli bacteria and inject heterologous proteins of interest, such as antibody fragments, immunogens, enzymes, transcription factors, or toxins. References -Piñero-Lambea, C., Bodelón, G., Fernández-Periáñez, R., Cuesta, A.M., Álvarez-Vallina, L., and Fernández, L.Á. (2015). Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins. ACS synthetic biology 4, 463-473. - Ruano-Gallego, D., Álvarez, B., and Fernández, L.Á. (2015). Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells. ACS synthetic biology 4, 1030-1041.
cancer DNA recombinant Probiotikum pathogens Pathologin molecule strains Strength Vaccine protein injection overexpression cells areas type cancer surface organizations cytoxicity Tumor properties Interleukin 7 genome antibodies Flunitrazepam systems function radicals inflammation cells coli Medicine modules
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surface regulation factors secret pathogens translocation Terephthalsäure strains specific chromosome van Strength protein cells proteins polymer proteins Inneren membrane
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polypeptide chain factors pathogens DNA man synthetic strains transcription factors protein coli cells sore type Salami surface polypeptide chain additional antibodies genome beam induced firm systems cytosolic synthetic Song of Songs metabolic pathway enzymes materials inflammation cells proteins selective
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okay so first I would like to sign the organizers for the disc an invitation and I will try to to move to pick your attention to the engineering we are doing to try to evolve or to generate microbes bacteria that could be used for tongue therapeutic applications so they are there our approach is let's say a modest one trying to generate modules that can be combined in say a chasis that of your choice and so it's not kind of a radical issue like complete engineering of the genome or changing the basis that are so we more focus on but in developing these particular modules so I will concentrate today in our work that we have been doing to provide a target in addition properties to E coli and also to provide the expression of injection to nonpathogenic echolight strengths so as you probably know the usual bacteria in medicine has been quite limited and certainly most application that you still see in the market comes from the use of probiotics naturally strained the has certain properties so they're considered natural so different joggers or front say certain isolates from a patient cetera they have certain strain darken that I consider beneficial and are given to certain port certain pathology to protect your hand your immune system and there are other mail application which is basically the use of attenuated pathogens to induce immune responses and then the produce vaccines are to induce vaccines or inducer vaccination since recombinant technology appears there was clearly interest in the modifying bacteria to produce molecules not only in vitro but also in vivo maybe to enhance these properties that are sometimes found in beneficial bacteria and in fact if you review the literature there's quite a few examples in which the people has been using traditional recombinant engineering to to modify probiotic strains or to modify known pathogens or even attenuated strains of pathogens to deliver certain molecules of interest like for instance interleukins or antibodies against inflammatory diseases signed cytotoxic proteins against cancer because bacteria are able to grow in solid tumor preferentially when they're administered systemically as you will see later and also other molecule that could intervene against infections and viral or bacterial infections so you could have a probiotic that could and protect your mucosal surfaces against this type of infection or even the lever certain molecules to cells in your in your gut so you can pre-program the metabolic sword 10 molecules produced by epithelial cells they're in contact with these microbes and this is our areas in which has been worked there's many groups that are working on developing this type of applications so well a
consensus let's say for us it would be to have total bidders with this synthetic biology you can generate modules modules of different types and and so ideally sin theta viola can provide a way to combine all these modules in a perfect chassis so you will not need to use a natural strain which what is partially beneficial but will have also other properties that are unknown okay so we could ideally think on introducing in our chassis studies may be indicated for a certain application all number of modules to sense the environment and response to that or for instance to by delivering of protein we can also introduce modules that would allow the bacteria to move towards certain signals so targeting the bacteria to certain areas or introduce reporters that could indicate us that the prison of a certain molecule or up or at the present of a certain disease and all could be contained by certain mechanisms of containment so you can introduce in your back ti all these type of things I would talk as I mentioned previously on a module that we have developed for targeting the specific addition of e.coli and to silts and surfaces and elated about our delivery module based on injected zones so why we
focus first in this type of modules to target the additional bacteria to certain cells because we consider that this is an essential property if you want to use bacteria for therapeutic applications is to to have the possibility to the leverage of bacteria to the type of cells that you are interested in or maybe in some cases you will have a pathological disease and itself that is inflicted by a virus so you have an antigen of a virus there or it can be a tumor cell and you will have certain proteins press but also in addition to this type of application you think that these modules could have other application also in vitro let's say on avoid a periodic surfaces so you can maybe develop this type of addison's against surfaces so so that you can help your bacteria to attach to a certain surface for instance in a biosensor
story I think it moves in the wrong
direction okay so if you see what's in nature and so what happened how the bacteria adhere to different surfaces is they have developed a number normal usually of proteins or polymers of proteins that are a symbol on the surface of the bacteria and that allows the bacteria to recognize certain structures on surface usually as any strain any natural strength courage multiple editions against different with different specificities and so it's difficult to you to reprogram a natural strain and if you do not delete partially the mayor additions that it contains another problem with natural additions also is that the specificity it is kind of broad and low and this is because a frequently recognizes carbohydrate and as you have heard from the previous talk carbohydrate are frequently found in many proteins so bacteria will have the tendency to bind to more than one tissue and my order so this one more than one cell types with using natural artisan so you have to really engineer something new if you want to be more specific and so we thought of just based on what is known about an additional such as i told you that can be anchored on the surface we play with different possibilities but we wanted to reduce this actually say to modular part one is a domain that will carry the addition property and the second will be a domain that anchors this to the surface of the bacteria so what will be the ideal properties of this type of domains will be high affinity and specificity of course and additionally that it will be something that you can select from the library okay so you can later just simply by changing this edition model you will be able to target two different surfaces so you will have a method of selection and a large library of this type of domains so we thought of using why not immunoglobulin domain because natural additions in bacteria frequently have ig-like domains like insane bria this is a female domain of a pill I type 1 filler in akola and it carries an ID like form okay so it's possible to use a human or other type of antibody like domains to replace for natural additions
and we decided to use after testing different types this type of antibody molecules that are derived from camel antibodies which carry all this specificity in a single domain of the variable V H domain these antibodies are are found in commodities they were discovered in in Belgian and by the group of search will determine years ago and this has it very interesting properties in binding and solubility etc and so there and one thing that attract our attention also was that they are simple and you can express them very well in in bacteria okay and in terms of
selection this system also allows to easily select the specificity the specificity you want I just simply summarize the standard methodology that has been livid that is developed by their other alternatives just after immunization of an animal of this type you generate a library of genes from the b-cells to plunder the nickel I'd you can infect with bacteriophages makeup will activate and select the specificity of the clone that is recognizing the antigen of your interest as is a simple scheme for basic technology that was developed time yes ago so we decide to use this type of antigen-antibody like molecules of sedition domains and we later of course code develop a way to anchor these domains on the surface of e.coli and with that is different ankle
domains I'm going to make the story very sure we the domain of the proteins that end us to be the best ones would were derived from interim and pathogenic ecoline dramatic equalized strains that are called in t means in t means our protein that are expressed on the surface of e.coli of these strains and allow the bacteria to attach to the surface of enterocyte it carries a viral a domain that anchors in the outer membrane and it displayed ig-like domains toward the surface and
this is shown here this is this but the outer membrane of the bacteria here is the hot cell and this bacteria attached to the enterocyte using intimates which display this ID like domains for contacting a specific receptor that the bacteria inject into the intera sites so it's a protein that has a certain specificity a clear specificity for a specific protein that is not found normally in in the mammalian cell so we
end up with this type of constructions with an outer membrane anchor domain and this antibody domain and we started to
to express these modules with plasmids and standard plasmid that you can induce in a strain that lacks the mayor fimbria type in e.coli so they do not attach to manos residues and we found that they were correctly espress display on the surface we introduced tax for the immune detection and that they bind correctly the epitope on solution and we also tested if they could somehow and arrives the addition of all the bacteria to to a surface coated by the antigen recognized by the antibody in this case so we coat a lie supplied with different antigens in Tower which we have nano bodies against them and as you can see if you
improve eight these surfaces with backtick with e.coli bacteria is pressing and this additions on on the surface you have bacteria attached to the surface of the plastic coated with a 15 odeon or gfp you have addition these additions recognizing gfp or human fibrinogen on the surface so we decided
to move from the plasmic to the chromosome to avoid antibiotics and to avoid the complexity of making induction etc so what we did essentially was to inter recombine our plasmid into the chromosome is justit of the P tag promoters by considered if promoters and delete add simultaneously that we introduce other reporters like bioluminescence in our strength we delete other editions that could be interfered with the audition process and
one thing that we tested and for us was very important was to see at the same time that we introduced this construct we remove all antibiotics okay but one thing that we tested is that not only the expression of our concern it was okay in the chromosome but also the growth and the viability of the bacteria was perfect so it was essentially growing at the same rate and it has the same by ability than the strain that doesn't carry this construct in the chromosome and as you see the expression of the constraint is maintained throughout days of continuous culture without any requirement of any selection pressure so it's just growing the strains in lb okay so we wanted to test this with cells and since we had this type of and certain antibodies that nano bodies against gfp and also against the translocated intamin receptor is an antigen of this effect which is not found as i told you before in in hela cells or in human cells and in any part of your body so we thought this could be nice examples to test the addition of these strains to tour cell so we make stable transfectants of HeLa cells that express on the surface the antigens recognized by the addition see in this case was gfp display on the surface anchored by an transmembrane domain and and this is the region of of this antigen that is still recognized by this edition and it was anchored to another fluorescent protein that is not cross doesn't cross recognize well first we as we say and unaware ASDF p and we tested initially growing cells in vitro on plates and the hilda and transfected cells or cell that expresses gfp on tortilla on the surface and we incubate these wells with our strains that one that is a control or the one that expresses it the addition zach is gfp or tier and initially the as i told
you before we introduce this value luminescence so you can see all wells that are producing light so and once you was to just see what are the bacteria that remain on the world's after washing you just see that only the bacteria that expresses the addition against gfp binds to hela cells expressing gfp on the surface and only bacteria expressing this antibody against still binds to the world that has carries a HeLa cells with tier on the surface you take a look at what is inside you could be able to see
it better so you will have cells this is a field in which you can see some untransfected cells and other cells that are expressing the gfp and this is a stain for e.coli and as you see ecoli binds to cells that expresses the GSP on the surface and the controller strain that recognize a different antigen will not bind to these cells ok and a nice control is this non untransfected don't respect the cells that as you see are clean of bacteria this is a more or less
the same but II with confocal microscopy selecting a feeling which you have a transfection and non transfected cells and and you can clearly see the number of atera surrounding your your target self and the same happened in the case of the other addition against tier you have a cell that is present here it's fully covered with bacteria so another
important thing is the speed at how this happens and we make a video to see how in in time in a time frame of less than three minutes of infection you have are here a cell that is non transfected and the bacteria go a scan a little bit the surface and then escapes and whereas in cells in which and there are expression of the target bacteria go and attaches to to the surface and stop moving in and this is something that of course that you see in less than three minutes so
it's very fast we wanted to test this a little bit in vivo and we you probably know bacteria especially anaerobic bacteria has been used since more than a century ago to try to eliminate tumors because certain bacteria specifically anaerobic can grow in anoxic areas in solid tumors and this is were that was initiated more than 100 years ago but as I said there's being their group like Neil forwards from from Michigan who are engineering salmonella strains and others to be able to deliver specifically and molecules of interest in the tumor one issue with this type of essay experimental approach against cancer is the dose that is safe to Viet minister of course you need a bacteria that is nonpathogenic but also you need low dose to avoid a systemic infection at the but at the same time that those has to be high enough so that you warranty the colonization of tumor by by the bacteria so we decided to test whether the expression of these glues this edition on the surface of the bacteria helps the colonization of the tumor and so we established with ourselves a model experimental model based on hela cell expressing gfp so you can do it by subcutaneous injection do a nude mice you can generate a solid tumor in the flank on the mice and later you can introduce your bacteria by systemic infection in the tail vein of the animal and it is reported that in this type of approaches with bacteria and colonizing tumors three to five days after injection there's a peak of the proliferation of bacteria in the tumors whereas other organ usually this is a half less less number of bacteria and as you will see and so we decided to test whether our bacteria were able to colonize this art tumors with expressing gfp and it would be a better targeting of our strains and initially we just simply use the dose that is reported by the people on that is used in salmonella or even ecoli head 12 to access a tumor a solid tumor that is in the range of ten to the seven and as you see both our strain that is targeting the tumor that expressing gfp and the strain that is a control colonize it equally well six out of six animals were the tumors of these animals were colonized efficiently and with a having a bacterial proliferation toward 10 to the power of eight per gram of tissue two more whereas other organs like liver and spleen we're almost clean of bacteria okay so that means that at a high dose there is no difference so we wanted to test what happened if we reduce the dose okay if we really a force a condition that is not optimal for the bacteria to colonize of tumor and so we did you see those two one percent of standard dosage in 10 to the five and as you see from this as light
now then there's a significant difference between the strain that carries an addition that targets the tumor in which in these animals eight out of nine animals were colonized the too much of this animal were colonized by the bacteria efficiently whereas in the controls only two out of nine or three out of nine weeks in the wild type strain that carries nonspecific Addison's natural string were colonized so this engineer strain significantly enhance the colonization at lower doses so you will you are able to target more efficiently bacteria using a lower dose to add two more and this is a control of a specificity we introduce a group in which the tumor were generated by HeLa cell that were not expressing gfp and in this case our strain was not able to colonize this tumor more efficiently was like in the control identical to the control so to
conclude a little bit this part of the talk I I'm show you this construct this edition that can be expressed constitutively from the chromosome of the bacteria and that can allow you to target specific cells or surfaces or tumors in vivo and I we believe this we are developing additions that will target real to Moores with specific markers and in areas in which bacteria may may access and may deliver a specific molecules toward the tumor cell or in the tumor environment so I then
we'll focus on one of these put a delivery machines machineries and as you
probably know bacteria had evolved a number of protein secretion system specially granulated bacteria to translocate proteins across the inner and outer membrane and one of these son of them are simple but many are tunnel others are strawberry complex and allow direct translocation from the bacteria to another cell than this another cell in for this particular type of protein delivery systems usually is a Imperial cell or a mammalian cells plant cell and so many pathogens from especially grand- estranged from pseudomonas or from equalised salmonella carries complex machineries that are called injecting sins for the tide free secretion system that allows the translocation of a specific proteins toward the cytosol of the infected cell and this effector proteins that are translocated by this machinery are atomic multiple functions in the hotel but helps the infection to proceed they can target cell cycle they can target inflammation they can target the cytoskeleton and mitochondria so they have multiple possibilities to to target and usually these machineries are coupled to a dedicated atps that energizes the system for for translocation they carry also the factors that are translocated carries a short n terminal sequence that is not cliff but is recognized by the machinery to be translocated tower the cytosol of a mammalian cell so in a way this is
like ash range a molecular syringe for translocation of proteins and thus create a an interest in using this type of devices to tour a specific delivery of molecules in the cytosol of mammalian cells so we decided to to try to
assemble this type of injection and based on the injected under express by enteropathogenic equalised strains which as I mentioned previously helped the bacteria this pathogenic strains to attach to the entire sites so they are syringe like complexes as I show you before but there is significant difference of this type of injection is that the it stands filament a very long filament that can be up to almost a micron long to access the enterocyte even from a distance so he can inject proteins not even with a close contact it can inject protein initially from from a distance and then it introduces for instance this transfer gated intamin receptor and then it binds intimately to the enterocyte later ok so these filaments have a channel in which the proteins are supposed to be translocated through this four of the of this monitor so this is a scheme of how
epic attaches and inject the translocated intamin receptor and form this attaching and effacing listenin to this particular interaction which in this particular case the tier is also able to signal in to signal and to reorganize acting on the surface of vendetta site and so the bacteria will be a less a sit on a pedestal of acting so it's something that is very easily seen is that the bacteria remains insta cellular but the acting polymerizes just underneath the bacteria having a leica that is the like structure bacteria not only introduced ear introduces as i mentioned previously many other factor that has other functions this type of bacteria but us so initially our interest was with antibodies but because as you know antibody molecules are usually targeting the cellular receptor or soluble antigen simply because they are accessible but there are many other targets inside the cell which are important and which are difficult to access using an antibody strategy so we decided to so we thought that since equal i can be used for selection of antibody molecules why not use the same strained also for delivery but as I said the this type of injection could deliver many other proteins of interest not only the effectors so we did some some work
with the pathogenic strain just to test that these syringes were capable of translocating small antibody fragments like the Nano bodies that I mentioned previously and as you see we tack the nano bodies with a sequence that is recognized by the way from a naturally factor and we could see the secretion in this case toward the medium specific dependent on on the type 3 the system of the antibodies fragments and we made different experiments to show that they were active they were binding the the antigen with similar affinity and these are just simply mutant in which you knock out the ATP a TPS and then without this a TPS activity you don't see any secretion of the molecule or other proteins that are forming this phenomenon the poor and we also test that this antibody molecules could be with this system could be injected into mammalian cells not only secreted with
the sitting with the system with its different experiment maybe the more visual is this one in which you use a reporter that is an enzyme that I lactamase that can degrade as a straight a fluorescence estrogens and infected cells of this what you see here are infected hela cells that are you don't see the bacteria which is simply see the HeLa cells and if the Vita lactamase is inside the cytosol of the cell then the this substrate of that is green and a beta-lactamase turns blue because of the enzymatic activity of the Beadle activate and so we use this assay to test whether we could translocate also the nano bodies with the several of them and we also see a translocation of fusion between the nano bodies and the enzyme in by the HeLa cell so we it was clear it
was clear that there was a potential to translocated Rose proteins using the injections of epic but the problem was that a peg is a pathogen so you are not going to use a pathogen at all for introducing anything especially because as I told you epic not only introduces the protein that you are interested in it introduces over 26 effectors other protein that you are not interested on so we decided that the best way to go would be to move the injected somes to a known pathogenic strain and see if we could assemble these complexes the complex delivery into a k-12 a strain generating something that we call us synthetic injector equalised stray cats eh just to continue with this epic a hug CX strength editor the nomenclature that a day sorry so how we introduce this
into k12 was so how we envision the introduction was not easy it was previously reported that you can clone the whole island into a cosmid and then you could cease an assembly of the tide three but it was very weak and partially because the regulation is not maintained and so the loss I that encodes all structural proteins of this inject Assam is located in that region of about 35 Clovis's that is called the locus of enterocyte effacement and it is it carries the structural genes required for the Assembly of this machinery but it also carries all the genes that might not be they are not interested for for synthetic approach like regulators or the sign of factors that are being injected and that you don't want to have there so we decided to pick from this loss I only the genes that were required for translocation and since we wanted to have a good expression and a controllable expression we decided also to remove the promoters from the are constructs and so we what we decided was to remove the regulators and remove the effectors and promoter regions and so we just decide to amplify the genes that were coding essential genes to assemble the injection in a functional way so we amplify this engineer operands without any as you say only the or open reading friends with no promoters no regulators and no effectors and we introduce this frames of fragments into vectors that carry a PTAC promoter to induce the system by addition of iptg initially and as we are not we don't want to to to work with plasmid or antibiotic resistance as I mentioned previously so we decided to integrate all our constructs in the chromosome a different position and in fact what we did was to integrate them in replacing natural additions found in bacteria so our strain was father deleted of natural addition factor and during the process of integration so we integrate these five operands in the chromosome replacing this cryptic Addison's found in k-12 and with indeed generate a different strain an additional strain that lacks the promoter in the first operand of the system so that we could have a control of the expression of a control of the specificity of the translocation of any protein that we wanted so the is an experiment that in which you can see growing the strain and comparing with the production of these inject isms in epic to compare it with the production in the engineers trained by a with addition of iptg and these are proteins this ESP de bien a are proteins of the filament that are in fact translocated through the inject ISM and for this filament to help to recognize the mammalian cell and then form a poor for protein translocation so once you see this protein is that the sky three system is active and as you see the three proteins be DNA are also found specifically when we induce in the supernatants of the engineered strain and you can recognize them by specific antibodies so we detect by western blot
different components but we decided to watch the father and try to purify the injectors ohms of our engineers strain and compared with what we could purify from enteropathogenic strain and as you see we could detect filamentous structures of different lengths that are compatible with the antibody under our cross and recognize a antibodies against this inject ISM and from a peck I'm from our engineer strain which are very similar in the loop very similar so our strain is probably assembling bona fide injected some of it back so we tested by
different methods how can we if our strain is able to translocate proteins into mammalian cells acting really as a syringe and since I previously mentioned
that the one that is more easy to test probably is the tier a factor why because one is the first protein that is translocated naturally by the pathogen it inserts in the membrane and then it polymerizes acting so these are simple readout to detect if there's been translocation of these protein by the tie three is that the acting polymerizes underneath the bacteria so we decided to to test whether our strength was able to translocate here and for that we introduced here with a specific a chaplain that is required for his secretion and also intamin now on the surface of the bacteria using amplifying this a fragment of the lee so we will have a strain that in addition to office pressing all the components of the inject ism will carriage this from the chromosome carries the the factor protein and intimate for specific recognition of tier and this is a
confocal image in which we compare the pathogen epic a strain in which i don't know if you are able to see something but the bacteria are staying in both cases with this sian color so it's what well what you see here are cells infected that do almost is difficult to see the acting because it's stayin in red and it's also hard to see but then you have to believe me that the bacteria this in this case is epic there's a small accumulation of acting underneath the bacteria okay this is a strain that last they are the epa's so in this particular strain you will see bacteria attached but there's no acting accumulation there's no red spots underneath and this are the engineer strain in which you see the bacteria in in Zion and then all this red is the polymerization of acting underneath the bacteria so meaning that our rack our engineering strain was capable of translocating this protein and into the mammalian cell in a functional form so i was able to signal acting polymerization and this is the specific control in which without the promoter you don't see any even bacteria attached to the cells in this particular case so these summarizes I
don't know what happened this is like a not so intense so yeah no no you can see
better huh yeah I don't know what was like a tire of projecting things so you well then you can see the image so this
is a summary of what we we have now but we are working on systems that may allow us to induce the system by not iptg but other inducer that can be used in vivo but essentially the the strain it is in a way in which we you can assemble this specific injected songs on the surface and then you come produce the protein of interest in a different position and then this pro team will be injected into the mammalian cell and so what could be the application of this especially we combine it with the targeting that I mentioned previously so we could deliver proteins specifically to specific cells and not only antibody fragment we can also think on natural factors found in in these pathogens this pathogen has like a catalog of proteins that 30 different pathways so you can choose something that act on for sale killing or for up in pain and inflammation for instance and enjoy you can and targeted particular pathways using this delivery of protein delivery in the cytosol of bacteria and of course you can introduce other type of things like ensign peptides for immunizations or why not transcription factor has been shown that certain transcription factor can be translocated through this system and can modify the genome also ave of the cell so I'm in this as an opportunity to to to do on real application with this type of material engineering so I would like to although you have been seen the people that has been involved in my presentation I would like to thank especially Carlos for his work in and synthetic Addison's valleys ballon Theo has developed this system also for selection so we can select against different targets the additions and the nano bodies that are of our interest using directory bacterial display system under which was mainly responsible of the engineering that have the tie three in k-12 and I would like
also to to thank a friend professor God Frankel for impaired college who's an esper in effect effect etc and who introduced me to all this wall initially and to Victor of course who has been my lesson mentor in the past and who is still aspiring many ideas and in our group and so thank you for your
attention and
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