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Synthetic Morphology Approaches in Pseudomonas putida for Bioremeditation of Haloalkanes

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Synthetic Morphology Approaches in Pseudomonas putida for Bioremeditation of Haloalkanes
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Bacterial biofilms are known to outperform planktonic counterparts in several types of whole-cell biocatalysis processes. The transition between planktonic and biofilm lifestyles of the platform strain Pseudomonas putida KT2440 (as in many other Gram-negative microorganisms) is ruled by a regulatory network that processes a large number of external and endogenous cues into different levels of the trigger signal cyclic di-GMP (c-di-GMP). This circumstance was exploited for the rational design of a synthetic genetic device that supersedesthe processes involved in synthesis or degradation of c-di-GMP in P. putida–thus making the bacterium to form biofilms at the user's will. In so doing, the transcription of either yedQ (encoding a diguanylate cyclase) or yhjH (encoding a c-di-GMP phoshodiesterase) from Escherichia coli was artificially placed under the tight control of a cyclohexanone-responsive expression system. The resulting recombinant strain was subsequently endowed with a synthetic dehalogenation operon (spanning two genes from P. pavonaceae encoding haloalkane dehalogenases) and tested for 1-chlorobutane biodegradation. Upon addition of cyclohexanone to the culture medium, the thereby engineered P. putida cells formed biofilms displaying high levels of dehalogenase activity. These results show that morphologies and physical forms of whole -cell biocatalysts can be genetically programmed while purposely designing their biochemical capacities. Furthermore, the spatial disposition of the bacteria at stake will in fact become an integral part of the design process of genetically-manufactured catalysts of the future
type synthetic biology man synthetic metabolic pathway subject Combining Magnetometer Altbier tool nickel yeast biochemistry selective
informatics synthetic sense cell genetic engineering plant pathogens scaffolds metabolic soil stress processes
Bohr los gene blocks genome enzymes Brown adipose tissue phosphate saline transformations surface Periodic acid-Schiff stain regulation rates Maische properties thickness chemische Reaktion NADPH polymer resistance sodium hydride Todesbescheinigung Pyruvic acid Second messenger system Polyurethane Ketone pathogens hydroxyl groups man synthetic strains Zeitverschiebung important Oxidativer Stress Butcher oxidation power morphology Biocatalysis Nicotin galena variability conditions metabolic pathway salts substrates Pentose Glucose Katabolismus platform metabolic Polysaccharides Humifizierung molecule model Oxalsäureestern biochemistry Dehydrogenase Redoxreaktion Xanthine oxidase concentration steps Deep sea Dihydroxyacetone genome source phosphorylation firm end Mauveine glycolysis medical phosphate orders of magnitude Gamma compounds form Toluol species Flagellum adhesins case aromatic fructose solvents Gluconsäure stress cell cascades control prosthetic groups biotechnology processes type Glucose solid-phase extraction Ketone mode carbon Pentose phosphate pathway synthetic initial Peroxyacetylnitrat Genregulation Bewegungen Angiotensin-converting enzyme AdoMet Biopolymere
Zellzyklus Plasmide gene cyclohexanone man restriction synthetic Revenue enzymes protein transcription control Vector overexpression soil cyclase tool Cyclohexanol biochemistry standards type chemical element steps slides induced metabolic pathway Optische Untersuchungen Genregulation standards substrates modules
type Polyurethane steps fluorescence Plasmide dynamics cyclohexanone induced systems lone pairs enzymes GFP Peroxyacetylnitrat control Genregulation overexpression salts
Glucose Digestion growth case Crystal violet cyclohexanone Bernsteinsäure strains lead cell Vector overexpression rate cyclase Cyclohexanol type Glucose surface Fucose concentration glass carbon Angular mil source induced systems glycolysis conditions GFP Bernsteinsäure coli
type sense phase surface morphology glass Plate Plasmide case Crystal violet stability induced man strains systems cellulose GFP Indicators cell control overexpression cyclase derivatives Sauce
sugar Transposon growth los operation gene case Einschnürung Unterdrückung <Homöopathie> cell transcription overexpression translation type power biosensors operation morphology regulation concentration EPDM stocks Phenylalanine ammonia-lyase systems conditions HPLC GFP Peroxyacetylnitrat Genregulation
Phenylalanine ammonia-lyase firm chromosome biosensors Transposon cell concentration Peroxyacetylnitrat Plasmide fluorescence Benzodiazepine level
Zellzyklus activities genetic code Alkyl Halides complexes chemical chlorine Phenylalanine ammonia-lyase Carcinogens strains Indicators model metabolic pathway race Clenbuterol soil protein synthesis biochemistry
stretch Dehydrogenase species Polyurethane activities Alkyl Groups Alkyl Halides growth Aldehydes operation chlorine formal enzymes cell soil biotechnology mineralischer biochemistry type Redoxreaktion operation Biogenesis Plate source Adenosine Mineralisation metabolic pathway compounds remove Butane
Bernsteinsäure Glucose activities cell concentration protein control salts Dielectric spectroscopy
physical chemist sense factors Humifizierung man synthetic enzymes cell tool oxygen processes biochemistry chemical element resistance organizations Anaerobic exercise morphology mode steps synthetic biology chemische Reaktion synthetic level enzymes variants Gamma oxygen
okay good afternoon everybody I would
like to start by thanking the organizing committee for this nice opportunity to be talking here in this very nice conference and today I would like to share with you some of our latest results on biodegradation using pseudomonas putida as a microbial chasis so now we will move from sciences which was the main subject or of Erika stock to via the gradation so i would like to start just by adding devoting a few minutes to what is the the path of a very typical synthetic biology persevere so what we normally do is to select biological parts and using genetic tools create new genetic devices that we plug in in a biological chap chassis so in the first part of my talk I would like to pay some attention to the selection of an adequate chassis for synthetic biology so we have heard so far that there are many types of microbial or biological chassis anyway and we have heard nice examples of yeast eagle eye and bacilli anyway today I would like to stress some of the interesting features of environmental bacteria as microbial chassis for bio degradation okay so if
we take into account the metaphor of cells living cells like computers making more computers one can think of a chassis like the structural scaffold in which one would in which one wants to implement genetic devices in the same way that the hardware execute the software in that sense what we would like to have in a nice micro lsac is a number of interesting features such as in the first place it has to be safe from a biological point of view that is that is to say it hasn't it shouldn't be a pathogen it has to display a very robust metabolism and some of the results i'm going to talk about really are closely related to this point in particular the chassis has to be very resistant to a stress either endogenous or exogenous in the vaio process and of course it will be very desirable that we can actually manipulate the chassis the way we want in particular in terms of genetic engineering and last but not least it has to be stable so that means that if we engineer something in this chassis it should be maintained like that for the longest period of time possible in this
sense in in our lab we advocate the use of microbial chasis derived from bacteria that come from natural environments and in particular we are very interested in using bacteria that comes from contaminated soils which for us is like a treasure trove for microbial Schatz's in particular we use an a bacterium that was isolated from contaminated Souls like 30 years ago which is pseudomonas putida and the
reason for which we like this bacterium so much is that it is naturally endowed with some of the properties I have been just discussing in the first place and very important for biotechnological applications pseudomonas putida is a non pathogenic bacterium and in particular the type strain of pseudomonas putida which is called Katie 2440 is not only nonpathogenic but I'll it also has been given the grass certificate which means that this chassis is generally regarded as safe for Biotechnology procedures on the other hand from 2002 we have the complete genome sequence available which means that we know all the genes encoded in this bacterium and over the last 25 years or so it has been used for a number of applications such as in situ bioremediation and the Indra industrial production of poly hydroxyl can wait which are biopolymers there are several companies that are presently using pseudomonas putida for biopolymer production but what is it very interesting as well is that since you can find this bacterium in heavily contaminated environments it is naturally endowed with a number of pathways for the gradation of very difficult to the great compounds such as aromatic compounds pseudomonas putida is able to degrade toluene saline and other aromatic compounds as well all these features speaks in favor of a very remarkable adaptability to thrive in very different and harsh environments in other words this bacterium has to be endowed with a very high metabolic versatility and in particular our main interest in the lab is to tame pseudomonas putida to be used for a number of applications as a first step in that and they were what we want is to be able to refactor the central metabolic pathways of pseudomonas and just at a glance it might seem easy but as you will see in a moment we discover some features in the central metabolism CEO of the ominous putida that where that are very unique and different when compared to other bacteria that are normally used as chassis in biotechnology so of course the ultimate goal here is to use it as a predictable biological chassis for bio degradation and biocatalysis so if one has a look at the central metabolism of pseudomonas putida as i said there are a few remarkable things to mention so in general if one thinks about bacterial metabolism one will think about the use of a carbon source to convert it into biomass and to produce energy out of in during this process and to generate reducing power in during catabolism a remarkable property of pseudomonas putida is that it can produce a lot of nadph the rate of nadph formation is you Dumanis beauty de is orders of magnitude higher than in other bacteria and this is one of the reasons for which these bacterium's is so resistant to stress produced by solvents and to exceed a Tiva stress and of course this is a feature that we want to exploit for biocatalysis purposes because NADPH is of course a very important cofactor in many biotechnological processes if one takes a closer look at the central metabolic pathways in pseudomonas putida you will see more or less this mess over here so let me why'd you through through the main steps in the Katawa in the upper catabolism of pseudomonas putida in the first place when cells grow onyx sources glucose for instance there are there are two possibilities for the processing of the substrate on the first place there is a phosphorylation pathway that starts with glucose and of course the first step is phosphorylation to glucose-6-phosphate but also under something very typical of pseudomonads in general there is an oxidation pathway that oxidase oxalates glucose intro gluconate or keto gluconate before phosphorylation so there is a branching point of xo's processing as a first step in the catabolism but a very remarkable thing about the central metabolism in this bacterium is that it operates the Ender do love catabolic pathway which is composed of just two enzymes edv and eda any it transforms 64 gluconate into to try OSIS glyceraldehyde-3-phosphate and pyruvate on the other hand another remarkable thing that makes ceremonies different from other typical bacteria such as equal I for instance on other inter bacteria is that there is an incomplete and the mayor of Parma's pathway the linear glycolysis typical of Eagle Eye the reason for that is that there is an enzyme which is missing here that convert fructose 6-phosphate into fructose 1 6 phosphate so what happens here is that there is only one enzyme which is gluconeogenic but then that enzyme that goes down here is missing so
with these features one of the first things that we asked was what was the central metabolism regulator in this bacterium I will not enter in the technical details in here but I will be happy to discuss with you this if you are interested but what we found is that there is a an important reason for which the linear glycolysis is missing and that is that this bacterium operates a cycle for EXO's processing and that makes it very different to most of the biological chassis used in biotechnology so in pseudomonas putida glucose is first phosphorylated remember that there were two possibilities phosphorylation or oxidation and after it gets phosphorylated it uses the end the ruler of pathway to produce to try OSIS but a significant part of the tri OSIS are recycled back to X OSIS so there is part of the glucose that ends up in lower catabolism after transformation but part of the trails are recycled back so the interesting thing about this metabolic architecture is that the three main catapult blocks described for bacterial work together because we have components of the pentose phosphate pathway than the daughter of pathway and am the mayor of paneth pathway and that's a good reason to explain why Pseudomonas doesn't have this step here downwards but there is a further consequence of this cycle here as you can see the reaction catalyzed by glucose-6-phosphate dehydrogenase produces NADPH so in each turn of this cycle this bacterium is able to produce one a knee deep NADPH molecule and in any case under conditions in which the souls are growing on glucose there is a catabolic overproduction of nadph but even more important and I will not talk about that today but it's an important feature of pseudomonas as well when the cells are exposed to oxidative stress the amount of carbon that is recycled vac using this metabolic architecture goes up to six to ten fold which which favors NADPH formation under oxidative stress conditions the important conclusion about this very typical metabolic architecture here is that it enables you dominas to live in environmental niche that are characterized by very stressful conditions because using this metabolism it can over produce the N dph it also needs to counter fate as stressful conditions so this is one of the reasons for which we are so in love with simonis Buda it has very interesting characteristic for to be used as a chassis but today I would like to focus in one of the trades one of the features of this species that we want to manipulate as well pseudomonas putida is not able to form strong biofilms the biofilms it forms are kind of weak which is in stark contrast as compared to other pseudomonas species for instance pseudomonas aeruginosa is known to form very strong biofilms that's not the case for beauty and there is a further thing the ability of pp You Dida to form biofilms ver eyes from isolate to isolate so is a trait that is closely relate to the particular isolate you are working with and of course for the reasons I will discuss in a moment that that was something that catched our attention we have heard something about biofilms so far but there are three important features i would like to to stress out today in the first place the initiation of biofilm formation is sunny stochastic process not not totally understood by the way of course we know that is a multifactorial phenomenon it doesn't respond to just one signal is a multi senior and regulation that ends up in biofilm formation and the intracellular regulatory network that finally results in biofilm formation is very complex as well and not totally understood and in the case of pseudomonas putida it wasn't studied in detail anyway but of course biofilms as we have heard so far are negative and negative gather in for instance medical setups but there are some conditions in which biofilm formation by bacteria can be advantageous and biocatalysis is one of the examples one of these setups why is that so because one can use biofilm add cut as catalytic platforms the reason for which we wanted to have bacteria catalytically active forming biofilms is that in the first place cells in biofilms show enhanced resistance external stress so we can further improve the natural resistance of ppt de if the cells form biofilms there is a large surface to exposure to to substrate which is of course important but there is another trade which makes catalytic biofilms desirable and that is that there is a very limited diffusion of metabolic intermediate since cells are closely attached to each older the diffusion of substrate and in metabolic intermediate is favored from one cell to the other because they are physically together as compared to planktonic cultures and on the other hand cells in biofilms are known to display very low salt to sell phenotypic variability so they are more synchronized from a metabolic point of view which of course in a biotechnological setup is very important so what we wanted here was to be able to reshape biofilm architecture using some genetic tricks that I will discuss in a moment and what we wanted basically was to implement a synthetic morphology approach for biofilms that is to say that we will be able to externally control the way in which the cells stick to each other and the way they attach to surfaces if one thing about this stochastic process that I was talking about for biofilm initiation one things on something like this like a very messy arrangement of cells just sticking together and sitting on a surface but what we want is to be able to change this situation to one more control situation in we through an engineering of the cell surface and rewiring the cyclic GMP dependent biofilm formation regulatory network we can get into something like this a situation in which planktonic cells can be told when and how to stick together and to sit on a surface for the sake of biocatalysis today I will basically focus on this part of our approach that was to rewire the cyclic DGM be regulatory network that ends up with biofilm formation in pseudomonas putida so just a few words we have heard in some previous talks about cyclic DTMB regulation but the biochemistry behind biofilm formation has to do with this secondary messenger cyclic the GMP so as we have heard before there are a number of signals that determine cyclic the GMP formation and there is a cascade of a regulatory processes that end up in very different phenotypes in bacteria when the intracellular concentration of cyclic the GMP increases for the sake of my talk today there are three things that we took into account in the first place upon an increase in cyclic the GMP concentration in the cells there is a decrease in flagella motility so there is the switch between planktonic to sessile state and there is an increase in the formation of Famer EMP lie and in the formation of EXO polysaccharides that increase this thickness of the cells to surfaces and other cells as well as we also we were discussing this
for a while there are mainly two mechanisms for cyclic d GMP turnover in bacteria proteins that have the GG vef motive rd1 allayed cyclases that produce cyclic DGM be out of GTP and there are other type of proteins that display the e al motive there are fought for the hysteresis that degrade cyclic the GMB into GMP so what we did was to identify two genes encoding a d1 l8 cyclase and a phosphodiesterase from ecoli to be over express in pseudomonas putida to control cyclic d gmb formation and degradation and therefore biofilm formation but before doing so and coming back to my my
first slide what we needed was to have once we had the biological parts identified these two genes from ecoli what we wanted with a genetic tool to control to find condo to get a fine control of the transcription of these two genes that result in the production and degradation of cyclic GMP in order to do so what we did as a first step was a standardized a new expression system that depends on cyclohexanol as the inducer so in order to to get this this expression system running what we did was to take some inspiration in other environmental bacterium which is acinetobacter John Sony which is a nonpathogenic soil bacterium that has an interesting feature that makes it very similar to see you mon us as well and that is that it can grow and degrade a number of different substrate among them cyclohexanol acinetobacter has a whole pathway for cyclohexanol degradation and what was interesting for us is that there is a girl transcriptional regulator and a promoter driving the expression of one of them science involved in this bio degradation pathway that have been identified in such a way that this regulator activates this promoter in the presence of cyclohexanone so we had these two elements identified to construct an expression system so we did we took the regulator and the promoter we standardize the promoter and the regulator in such a way that it fits in what we call the standard European vector architecture which are a series of plasmids constructed in Victor's lab in which all the the enzymes are known the restriction enzymes unknown and all the modules of the plasmids can be interchanged as needed so what we did was to transform these that comes from a natural bacterium into an expression system that fits in into the standard European vector architecture this is
what we did this is the resulting
plasmid and what we did first was a very short and very quick characterization of the dynamics of the system using a fluorescent protein as you can see in here in the aves in the absence of cyclohexanone as an inducer the regulator and the promoter are completely shut down so the dance the response of the system is very dependent of on cyclohexanone when you induce the system and as the as you culture the salts in the presence of cyclohexanone there is some more or less linear response of the promoter up to we're getting a 30-fold increase in fluorescence after four hours so that was the type of dynamics that we wanted for the expression of this enzyme stud control cyclic GMP formation and biodegradation so the next step was to
use this genetic device to overexpress those ensigns from E coli in beauty this is a very simple experiment in which we while lead that we really measured biofilm formation using the crystal violet aside in a peep UT the strain that karai's the empty vector the vector with the whiny late cyclase and the vector with the one elite cyclist induced by cyclohexanone as you can see there is an increase in the amount of cells that can stick to the glass surface but of course we quantify that and as you can see in here this is the quantification of biofilm formation in a strain that karai's the empty vector and in a strain that carries the vector with the d1 allayed cyclists as plotted as the concentration of inducer used to activate the system as you can see in here as we observe with the fluorescent protein there is a more linear increase in biofilm formation upon addition of cyclohexanone this is some glucose as a carbon source but more or less the same situation happens on succinate so we can induce biofilm formation either under glycolytic or gluconeogenic growth conditions and it can be externally controlled so in the in the situation in which we induce the system would five mil amores cyclohexanol we got a three-fold increase in biofilm formation which for pseudomonas putida is a huge increase so
this is these are the results with the cyclists but we also have a digester ease that degrades cyclic GMP so what you have in here is more or less the same situation in which we tested biofilm formation by cells carrying an empty vector the cells carrying the same regulatory system we engineered but in this case driving the expression of the digester is so there is of course a decrease in biofilm formation irrespective of the Galman cells and just to illustrate that this expression system is tightly regulated we repeat this experiment with another expression system that we already had in the lab they'll act like i QP TRC system and as you can see as compared to the newly engineered system this or one is more leaky so now we have dishes see defined
now we have the part now we have the genetic device to overexpress this part at the users will so what we did was just to combine everything in the chassis in this case we use a derivative of a strain Katie 2440 that has been deleted of all the profits so that results in a more genetic stability in the chassis and the ability to propagate plasmids more easily than in the wild type strain so what we did in this case was to tag the cells with a fluorescent protein that is constitutively expressed so we can follow individual cells and we transform these cells with the expression system driving the expression of ye DQ which is the deewani late cyclase what we did in here is to grow the cells in liquid culture and to submerge in the culture a coverslip a glass coverslip to see how cells stick to the surface in the first place of course we run the control with an empty plasmid then we run the experiment with the genetic device for biofilm formation and induced and in here you can see that when the system is induced you can easily detect micro corner colonies on the glass surface so it is really working it is really affecting biofilm formation not only from the microscopic point of view in the sense that we can detect it by crystal violet but it also forms micro Connolly's on a glass surface what we wanted to see next what
was were what were the reasons for this microscopic and macroscopic behavior so what we did was to stay in the cells with Congo red which gives an indication of exopolysaccharide formation and cellulose formation by by the cells in here you have the sauce transformed with empty plasmid this is the morphology you get in Congo red plate and then us that have the deewani late cyclists induce as you can see the the colony gets a completely different morphology with this these blobs over here in the colony that can be also seen at higher magnification and in the same cells
overexpressing the phosphodiesterase you see a more blurry morphology in the colony border when we quantified exopolysaccharide formation we saw an increase of exopolysaccharide formation in the cells that have the deewani late cyclist and of course we saw a significant decrease in eps formation in the cells that overexpress the phosphodiester ease but of course all
this was just an evidence that the actual reason for the phenotypes was cyclic the GMB formation or degradation so what we wanted was to quantify it in a way and as we heard yesterday in Ming stock it is quite difficult to quantify cyclic the GMP in the case of Silla moniz one of the reasons is that the concentration of cyclic GMP peaks at a point during exponential growth and then it goes down very rapidly so it's very difficult to detect it by hplc ms and we we tried a number of different protocols but we couldn't so we resorted to the biosensor so what we did basically was to take some inspiration of a regulatory circuit that takes place in pseudomonas aeruginosa in pseudomonas aeruginosa this operon hear the bell abcdefg open encodes olden times needle for a particular type of exopolysaccharide formation which is the pail exopolysaccharide this system this this jeans over here are repressed by the flick you transcriptional regulator which is a fist like transcription rule later and it represents crip shin of these genes so that it blocks X supporting saccharide formation in pseudomonas aeruginosa but in the presence of cyclic GMP this repression is relieved this operon gets expressed and cells produced exopolysaccharide so what we did was to take the promoter that drives the expression of Pell a as a promoter to be using a biosensor to detect cyclic the GMP formation so this is the genetic device we constructed is at t + 7 transposon that contains the Palais promoter this one from pseudomonas aeruginosa and of course it drives the expression of a fluorescent protein there is a translation and coupler here to enhance the translation of the the construct and what we did necks of course was to test it in cells
that overproduce either the day one a late cyclist or the phosphodiesterase so
this is the level of fluorescence one gets when this T and Sevet rapti and seven transposon quraían the biosensor is integrated in the chromosome and when the cells karai the empty plasmid so this will be the the normal condition and this fluorescence correspond to the normal level of cyclic GMP when cells
were transformed with the diwani late cyclic sorry the phosphodiesterase the fluorescence went down indicative of a lower concentration of cyclic GMP and
there was a significant increase in the scene yell when cells over express the deewani late cyclists so that was an indication that we were actually modifying the turnover of nucleotides in pseudomonas and so that it produced more biofilm for that reason now we had this
strain engineer to produce biofilms and what we did is to use it for bio degradation purposes and the model we decided to use with haloalkanes this is a very complex family of contents but all of them are very toxic and one of the interesting features of some of them is that they are Santa biotic so they were introduced in nature after the the human race started to produce them they weren't they weren't there in nature before industrial activity at all and as a consequence of that there are very few degradation pathways that can be found in nature for these compounds the component we use us as an example and as a model is one chloro buting which of course is a sinner biotic and issues a lot in dry cleaning it is produced more than 50,000 tons per year worldwide and the thing is that of course is a very nasty component because it's potentially carcinogenic and it's very resistant to biodegradation there are no no means to remove one chlorine from soils for instance which is the place in which it ends up fortunately we were able to
identify a bio degradation pathway from a pseudomonas species not surprisingly the associa- as well so this this species that was isolated from contaminated soils has a pathway for one chloro butane and other haloalkanes biodegradation and complete mineralization but the thing is that the this natural strain that is able to the greatest growth very slowly in most of the let's say biotechnological setups and industrial setup so it's not a very good chances to work with in bio degradation procedures but it is a good source of diello genesis of all these compounds and in the case of one chloro buting there are two de ello genesis that have been identified that can be used to degrade the compound so what we
did was to construct a synthetic operon using these to do Genesis and what we did next was to combine all what we had so far so we got the chassis we transformed it with the genetic device for biofilm formation we also transformed it with this haloalkane degradation open with enzymes from pseudomonas pawan ASEA and what we did was to grow it in multi world plate and after the cells establish a biofilm we expose them 21 chloro bewteen and after 48 hours we remove the planktonic cells and biofilm cells and we measure the DL agenus activity and the removal of one chlorine and these are the results of course this
is the control in here you have the planktonic cells growing either on succinate or glucose this is the activity ratio normalized to the total protein content and what you see in here
is that most of the DL OJ's activity was recovered in cells come from biofilm so the cells in the biofilm were not only catalytically active but they were more active than plugged on excels in such a way that more than sixty-five percent of the total de la jeunesse activity was recovered in the biofilm cells moreover after 48 hours all the glory within 0.5 millimolar was completely removed in this setup this looks like a very low concentration 0.5 millimolar but this is the highest concentration you can use you can expose yourselves to without damaging the salts so this is the maximal concentration they can tolerate but after 48 hours they can degrade everything so I hope I have convinced
you that in this example we follow different steps in a way that in the first place with a sign it microbial chassis that can be used for bio degradation we also contributed a new synthetic biology tools that can be used for manipulation of the chassis but a part of the metabolic engineering of the ensigns thence imac tivities that are involved in bio degradation there is a new twist that we think it has to be taken into account which is the synthetic morphology approach to modify the physical arrangement of cells in a bio degradation process in order to get a superbug okay so this is an approach that we think it will become an integral part of a biocatalyst design in the sense that normally we are used to use planktonic cells in bio processes for industrial processes right so we think that synthetic morphology the way that sells really are arranged in a three dimensional pattern is important as well so there are a few things that I would like to remark just to end up there there are a number of approaches that we want to follow now that we have this this preliminary results let's say we would like to use stronger cyclists and signs we know that the deewani late cyclist from cowl of auditory centers is a more active enzyme than the one we use from ecoli we would like to be able to switch the cells back to the planktonic state after they establish a biofilm that can be useful in a number of setups and finally since cells in biofilm are normally subjected to oxygen limitation what we are actively working at is to change the lifestyle of pseudomonas putida pbut de katie 2440 is an obligate aerobes so we are trying to identify the elements needed to change that lifestyle completely dependent on oxygen to an anaerobe that will be very useful in biofilm setups in which oxygen can get a limiting factor with that I would like
to thank the financial support by the medical reactions and the European will require by organizations and of course I would like to thank the people who actually conducted experiments ilaria and encourage way so in the lab with this I will end up here and thank you very much for your attention