SynBYSS seminar with Prof. Beatrix Suess at TU Darmstadt, Germany, and Dr. Shrikrishnan Sankaran at Leibniz Institute for New Materials, Germany
Okay let’s start good morning afternoon and evening and hello all welcome and thank you simis people for joining us the 100 15th seminar and I call it a tu dumad day today is a special day called a liap ear day once every four years we have February 29 making a deep year
366 days instead of 365 days if you are born on February 29 you are extra special although your birthday happens once every four years well every day is special as long as we enjoy every single second and every single day of our lives today is the day when a person who died
Yesterday nearly wanted to live let’s do something special today and every day okay it is my tremendous honor to introduce very briefly our Pioneer speaker on this special day professor betric susus she is a professor at T damad Germany and an expert in RNA synthetic biology I visited Germany
Four times last year alone thanks to her kind invitation it was a wonderful experience meeting great professors and students at her University I want to thank her again for her hospitality and kind invitation at that time patrix thank you so much for your service to the community excellent research
Especially on RNA synthetic biology and also dedication to education the virtual Podium is all yours now and thank you again yeah thank you again I think uh for the kind introduction and the the possibility to to attend your your excellent uh yeah s this medum uh
Meeting here and I I wrote down I have to send birthday greetings to three friends because I really have three friends that have birthday today so um yeah and uh let me share my screen I have only a few slides I don’t want to talk too um too much and and too
Long about my uh my work I want to give a briefly overview what we are interested in what we are doing give one or two examples so let me start with this uh nice presentation about um RNA so each RNA um normally in the textbook you find
An RNA as a single stranded um um yeah sequence as it’s shown on the left side here of the picture but this is um only um partially true so AR a really can fold into highly complex structure and and by chance 50% of each single stred RNA will look
For a partner to to form form um double stranded region and this results then into this um to this yeah complex um um RNA 3 structure as you can see on the right side and these um 3D structures they can they are comparable to proteins they can form catalytic centers but they
Also can form binding pockets and here this is an example of such an RNA that binds a small molecule you can see in red this is topamine this is an abdom um soall um binding RNA that can recognize this um small molecule with very high Affinity in the low nanom
Range and if you have such an RNA that can bind a liant like a small molecule but also a protein then it is called abdom and if the cool thing is that such RNA abas you can not only find in nature but you can select them the noo and this
Is a method called select here you start with with a huge pool of let’s say 10 to the power of 14 15 different RNA molecules and each one forms into another shape into another confirmation so by chance if you want to find an RNA aboma that binds your target there is a
Really good chance that among the 10 to the power of 15 there is one so here is the the scheme of a celex you incubate this pool with a tar Target and then you can fish the the ones that can recognize your target you can reverse transcribe
It amplify it R uh yeah repeat this cycle over and over again and if you are lucky you will end up with this little guys here and on the left side you have an abdomen and uh in shown in Red AR shown red that binds for instance a
Small molecule as tetracyclin is shown here or you can have an abdomen that recognize prot in here you um um have the as a Brin the head repressor t r and an abdom that recognized and what we are doing beside that we want to find this abdom and we like to characterize them
We want to use them as tool as switches to control chin expression and here is a kind of overview slide what what we are doing in our lab we use these little tiny abdom here these guys and we we use them to control chin xersion either we
Insert them in the five Prime utr and then we can um stop the scanning ribosome so in the presence of the L and it’s structured is more more stabilized it can stop the ribosome and um the ribosome can read through if the abdom is with out it’s it’s like and or you
Can block the excess of the ribosome on the left side this you can do bacteria you can control splicing and you can control the MRNA stability so these are different um different um areas we’re working with we understand ourself as tool developer um we work in different
Organisms so we are not focused uh to to one um one one special biological model system but we want to develop tools that can be applied by a lot of different persons and here is only one example I want to show you here um there’s an abdom I
Already introduced that binds to Ted R here’s the RNA here’s the protein and with this abdom we could control splicing you see here on the left side you have two axons the one Exxon very short only the adg and on the other side is gfb and then we insert the abdom into
An in one into the infront and here the cool thing was there was a crypticus Spide within our abdomen and we have the other three SP side here and then we could change between the usage between the one and the other Spide so if the protein binds one spite was used if the
Protein does not bind and this is the case if we add doxic cyclin then it cannot recognize the abdom any longer then the other sply side was recognized so we could um distinguish between the one and the other um splicing decision and by exchanging here a part of the
Abdom into a nuclear localization sequence we could then decide if we add a nuclear um localization sequence to protein or not and you can see the results here on the left side on the bottom if we add the nuclear uh nuclearization sequence this means if the first supply
Side is used then all the gfb is in the nucleus almost the gfb and if not it remains in the cytoplasm so this only one example of such switches we develop and we have in the meantime lot of different abdomens that respond to different proteins to to different small
Moes and with this I’m already at the end of my short presentation here on the left side you see our favorite Pets the abdas and then the the select method and we use them to develop Riv switches and you also we in the last time we we
Started using them as bio sensor so here you can see our first attempt to to to create such letter flow as it’s it’s not easy but um we are in a good way and I hope that um yeah we can hear also develop uh valuable tools okay with this I’m at the end
Thank you very much for your attention and um I give over to um chair thank you so much wonderful excellent talk uh as you know I mean you know we are now Rance of RNA engineering you know because of multiple development in the field including crisper system RNA covid vaccine and I believe
You know RNA engineering will flourish more in the future so many people working on right now RNA and then you basically Pioneer you know your epom RNA engineering many many many decades ago so I really thank again thank you again for your contribution to the fear thank you you’re
Welome okay now the main speaker of today with our longer introduction Dr shishan Saran uh short Shiri is a research group leader at the night needs Institute of for new materials inm in Germany he has the bio programmable materials group that explores a young multidisciplinary field combining synthetic biology and
Biomaterials it focuses on the development of materials with genetically programmed functionalities capable of biosensing stimul stimuli responsive long-term drug release and manipulation of cell Behavior synthetic body tools are used to program proteins and probiotic bacteria to perform smart and beneficial functions this engineer biological entities are then incorporate in developed polymeric matrices resulting
In composite materials with highly ver vertile functionalities a wide range of tunability and insitute controllability he did his PhD at the University of 20 TW in Netherland he did his postdoctoral work at The curan Institute before he became a group leader he established his independent Junior research group in
2020 uh during pandemic I guess focusing on the engineering of probiotic bacteria with therapeutic capabilities along these lines he’s a scientific coordinator of a regional life needs science campus Consortium working towards the uh development of living therapeutic materials he has also been co-organizing the International Conference on engineer living material since
2020 which conducted almost every year from this all kind of achievement and activity you know you will see he’s a true Rising Star importantly thanks to him I also visited Germany last year to give two lectures for for a summer school and in the spring seminar at his Institute last
Year sh thanks so much for your invitation last year and also for taking time today please take it away it’s all yours now thank you yeah thanks a lot tesak that was a wonderful introduction um let me just share my screen quickly there we go wonderful wonderful thank you so um as T
Very nicely put uh I’m coming from the inm the live nits Institute for new materials and we in sbren in Germany what we like to say the heart of Europe and we do a lot of very multidisciplinary type of material science research you combine opo Electro bio elements together but that doesn’t
Really explain what I’m doing here at synbus talk giving a talk on synthetic biology um so let me try to justify that first so our work is based in the field of drug delivery which is a very prominent field within Material Science and the traditional way of dealing with
Drug delivery is to pack material so you take a material that’s porous or or kind of a gel and pack it with the drug that you want to release within the body and then once you put into the body the drug is released in some kind of passive or
Active manner so this this works out quite well for many diseases that need long-term drug delivery within the body but it does have certain limitations uh that that the field is trying to overcome and uh some of these are that you have a limited drug Supply so
Whatever you put in is all the drug that you can release if you have a small drug you can put in more if you have a big complex protein type drug then you can have less and then the amount of drug that you could release is less you can
Only have once you prepared it um in in most cases you can only have a monotonic release profile so whatever drug is in there it’s going to be released at a certain rate uh over time and it’s you you basically can’t turn it on and off when when you need it
And then it’s also quite unsuitable for complex of fragile drugs so as I said bigger proteins or fragile drugs um if you put them in and they have to stay there for a longer period of time they might degrade especially within the conditions of the body and then yeah
They they remain inactive so to to address these limitations uh we’ve taken a different approach we’ve taken a living approach instead of packing the materials with the drugs themselves we’re going to pack them with biofactories that produce the drugs in our case these are bacteria and bacteria are commonly used in the Biotech
Industry to produce many Pharmaceuticals and so the bacteria can basically live within this material produce the drug and release it outside the great advantages from this is that you have the factory producing the drug right within so you have a self replenishing drug Supply and these bacteria can live off the nutrients in
Your body and so you can in principle have an unlimited drug Supply within your body um you can engineer these bacteria so just as berrix showed quite beautifully with RNA there are many different ways of engineering the bacteria with switches such that you can control what is being produced and
Released over time furthermore you don’t have to externally produce purify and package the drugs um in Within These materials and then use them so you kind of reduce the costs on all those processes and so make the delivery of even more expensive drugs rather affordable and this method is
Particularly friendly for the biopharma industry with which is rapidly growing uh within the Pharmaceuticals all right so this concept uh is what we call living therapeutic materials which is the title of my presentation and I’m going to be talking about these hydrogen confined bacteria for smart drug delivery these
Living therapeutic materials um the concept is kind of like this we have the bacteria within the material we can either put it on the surface of the body or implant it within the body where they will survive and they will will produce and release these drugs my work specifically focuses on the synthetic
Biology aspect the engineering of these bacteria to produce the drugs ideally in response to external stimuli or stimuli from within the body so that it’s controllable the dosage of the drugs that can be released but this concept of engineering bacteria for drug release at least in the synthetic biology Community
Is not really new people know it as living therapeutic it’s been going on for a couple of decades now and there have even been clinical trials that show that using bacteria for drug delivery within the body is is very safe there’s been multiple clinical trials actually
Showing this but why don’t we have these uh therapies yet because it’s actually very difficult to prove efficacy it’s difficult to control colonization of the bacteria so when you have a disease in your body that’s localized these bacteria have to go and colonize there grow to a ideal population that can
Produce and release enough drugs and then uh cure the disease but from a Patient to Patient and disease to disease the condition it’s very difficult to be able to control how much of this bacteria can colonize a disease site and this is where the encapsulation comes in so the
Encapsulation on one hand actually does protect the bacteria and host from each other so it protects bacteria from the immune system of the host and the host from any um Escape of the bacteria into other tissues where you don’t want them and secondly it controls the bacterial
Population size so we do quite a bit of material engineering to control that there’s a defined size of bacterial population within which means that will Define how much drug is going to be produced by those bacteria and so we have much more control over the drug doses that can be released within but
For this talk I’m going to focus on the bacterial uh engineering and especially the synthetic biology in this perspective we’ve actually cloned uh or engineered many different types of bacteria to produce different types of of kind of therapeutic compounds we particularly focus on two strains which is eoli and lactobac
Plantarum right now and I will get to why lactobac plantarum but Eola is particularly used because of the genetic toolbox that’s available for us to be able to engineer it with different genetic switches and so on so I’ll give you a few examples of what we’ve done with eoli the first one
Kind of encompasses the range of research that we do within my group um and that is the the that example is about making a light regulated proangiogenic living therapeutic material so you can control the bacteria by light to produce a drug that induces blood vessel regeneration and this is
Applicable for wounds and and cardiac issues within the body now for this we engineered the bacteria with a very welln um optogenetic plasmid called pedon and what pedon allows you to do is whatever uh Gene you clone within this plasmid it can be produced only in the presence of
Blue light so when you irradiate blue light it gets produced and if not it doesn’t so we particularly made a fusion protein which you see down here which we call as ycq the most important parts in this are the yef which allows the protein to be secreted out of the
Bacteria we have a collagen binding domain that allows it to bind to The Matrix within your body which is largely made out of collagen and then it has this tiny peptide at the end called qk that induces that acts like a growth factor to induce endothelial cells to
Undergo this kind of new blood vessel regeneration or angiogenic differentiation so we had we engineered this this plasmid we put it into the bacteria we could see that when you shine light It produced the the the this protein and it released it next we encapsulated the bacteria within a
Secure hydrogel Matrix so what you see down here the hazy part is where there’s bacteria and that’s surrounded by a protective bacterial shell and below is just a glass cover slip which helps us uh with the analysis and the microscopy that we did in the study so you have
This kind of protective living therapeutic material right now and the bacteria within it when you shine light it produces and releases the therapeutic uh protein and this is what you see in the graph below uh when you are in the dark basically there’s very little production when you shine light it
Starts producing and then dark again the production goes down so you can control the production and release of your drug now to see whether this drug is actually active we take it into a miman cell uh kind of invitro model where we have a collagen Matrix so the protein has a
Collagen binding domain and so the protein binds to this collagen Matrix the collagen Matrix mimics the the your extracellular Matrix in a wound for example and then when we include endotherial cells there they undergo this kind of angiogenic differentiation so what you see in the images below is
That normally without this protein what you see is what what is shown in the do these cells form a uniform layer but in light when the protein is produced the cells actually undergo this kind of network formation a change in morphology and so it proves that the protein is
Actually active so this is an example of the kind of um synthetic biology and a bit of material engineering that we do to make these living therapeutic materials now another example we actually made bacteria produce instead of a growth factor and antimicrobial peptide uh in response to temperature
And this peptide is called darobactin it’s not a regular peptid it’s called a rip or ribosomally produced post translationally modified peptide so it’s getting produced and then there’s a bunch of enzymes that modify it into this potent form that allow it to kill pathogens like pseudomonas arenosa with
Um uh mic of around 2 microgram per ml so normally these this darobactin there’s a lot of research going into producing it the idea is that it’s a new antibiotic that can uh overcome antibiotic resistance problems and eoli is largely being engineered to produce this and the standard circuit that is
Used to produce this is an ipg inducible circuit so what that means is that the genes for darobactin the peptide and the enzymes that are required to prod produce it our our engineered such that the peptide is produced only if you add this little molecule called ipg which is
A kind of sugar basically there is this t7 RNA polymerase which you see in the Green Arrow which actually drives high level production of theat so when you put this circuit in eoli it starts producing ipg at levels above this miic so we get over 2 microgram per ml but
The switch is not really good so if you you see when this when in the absence of ipg you still have a high level of leaky expression our interest was to get this bacteria in the living therapeutic material so that it produces the zob
Active D into the body and to be able to control it we can’t think of adding uh uh sugar all the time and so we went ahead with trying to make it thermoresponsive so in response to a temperature change in the body and a temperature change that could indicate
Like that there’s a fever in your body so we took this circuit which is also very popular produced uh developed by Mikel Shapiro who was also in one of the synbus lectures in the past um so he made this really nice thermal inducible circuit where you have TPA which is a
Repressor that under goes diarization below 37 degrees and blocks the T ptpa promoter but above 39 degrees it sharply under goes D dimerization it monomerized and then it basically allows ptpf promoter to be active and produce autoactive so it works it’s also a little bit leaky but if you see the Y
AIS here quite carefully then you’ll see that we are 10 times lower than what we need to produce so somehow the promoter is not very strong and the response also you can see in terms of trying to inhibit pseudomona salosa uh you see that if ipg even with or without you
Have inhibition with ipg you kind of completely inhibit the growth of the pathogen but in the temperature responsive case at both temperatures U at 37 you don’t have any inhibition at 40 available so we needed to rectify this we saw what are the best parts of these circuits so it’s the
Thermoresponsive part in the the tlpa circuit and then the t7 RNA polymerase that drives very strong expression we combined these and we made a thermo amplifier circuit and what the circuit does is that the tlpa so the tlpa drive the production of the t7 RNA polymerase and so at high temperature t7 RNA
Polymerase gets produced and at that point it drives really strong production of doac and actually even to our surprise we saw that this not only produces enough darobactin to inhibit the pathogen but it also kind of vanquishes the the leakiness so we saw pretty much almost no leakiness or
Barely any detectable leakiness at 37° Centigrade but at 40° enough to inhibit the growth of the pathogen and this is kind of what the actual assay looks like so you have the murky uh media which is where pseudomonal genosa is growing under all conditions except for the
Point where we made our bacteria grow at 40° and that media doesn’t allow the pathogen to grow anymore so we have a bacteria that produces the darobactin at 40° but not at 30 s now a third example the last one that I’m going to show about eoli is an
Example what I like to call turning straw into gold like the rumus fkin story um where we engineered the bacteria with two enzymes that convert a very cheap food grade precursor into a high value flavonoid so you can see this Che cheap food gr precursor camic acid
You get a 10 C cents per gram with the flavonoid costs about 7 ,000 per gra and so this bacteria is a probiotic nle 917 so you can actually eat it and the idea is that if you have it in your body and you consume cinnamic acid it’ll convert
It into this flavonoid directly and so it just took two enzymes that to do that um we were able to achieve the the production of this or this conversion within the using the bacteria then we were able to encapsulate it in a similar kind of hydrogel con that I showed
Before so again the bacteria in the center they envelop by a protecting shell and we could see or you could show that basically with different amounts of cinnamic acid that you add you could control the amount of pyosin that was produced and also we were able to show
That these kind of constructs could basically the bacteria survived for a long time for at least a month and could keep maintaining a decent level of production or conversion of cinnamic acid to pin through throughout this time so these were the examples with eoli but then we we always have this
Question is eoli really the best bacteria for this technology I mean it’s it’s great for making genetic circuits you have so many genetic Parts but its applicability is sort of limited to the gut largely it’s kind of native to the gut and you put it other places in the
Body you can cause some kind of reactions and problems so we started to look around to see okay the microbiome is present in many parts of the body what are you know like a family of bacteria that are present in all these places and we came across lacto vasili
They’re either friendly common cells in all of these uh organs in the body or they’re used as probiotics to um to actually provide some kind of B health benefit uh in in these organs so that was great okay lactobac is something that we could engineer is what we thought but there were many
Challenges there the first one was which species do we use the family of laob basilaia uh is is so huge that in 2020 they split the family uh into 26 Gena at that point um there were more than 200 uh uh strains within it and all of them
Had different types of effects in the body so we actually looked through the literature and we saw there was one particular strain called lacti plant vasus plantarum which was the most reported the most studied and the most engineered also although the engineering was very modest but the strain also was
You know had a unique feature that’s nomadic in nature which means you actually find it in a lot of environments uh inside your body and also outside so you can find it in fruits you can find it in vegetables you can find it in fish you can find it in
The soil you can find it in your body you can find on the skin in many places and it can adapt to surv surv in all of these sort of places it’s almost always only found as a beneficial organism in whichever host it it exists so that was
Wonderful um but then we had to come to the point of actually engineering it and this is where we Face many challenges first of all getting DNA in so for everything that I talked about engineering you have to get the DNA that we want we programmed all these genetic
Circuits into the bacteria we need to find strong inducible promoters so I talked about tlpa ipg all of these things well they these tools these parts didn’t exist in lactobac in this in this strain and in most lactobac and then producing and secreting therapeutic protein was also very limitedly
Understood and and and known and finally having antibiotic free uh plasmid retention because most plasmids require antibiotics within the body to maintain but or within Biore reactors to maintain but we don’t want to have that when we want to go for a therapeutic application so there are all these challenges and
Many of these we’ve actually uh come about solving but I will only explain or talk about a few of the things that we’ve done right now and you see the QR codes in almost every slide so you can you can check out the papers you want some more information or just contact me
Directly so the first one is getting the DNA in and what is normally done um is you you you kind of assemble your DNA so you I talk about all these parts and all these genes and so on so what we do is we get all of these genes together we
Assemble them together and then we have to put them inside bacteria ecoli is really happy to take these DNA so what people normally do is put the DNA into eoli and then they make eoli produce much more of that DNA take that DNA out and then put it into El
Plantar but we Face problems that there were some incompatibilities between the two certain genes that we were trying to engineer were incompatible with eal maybe the metalation patterns were incompatible so we ended up with mutations quite often when we tried to go this route now the main purpose of
Putting it in eoli is to get a lot of DNA because you need a lot of DNA to get past this thick cell wall of gr positive lactobacilli to get the DNA in and so uh Mark and my group basically had the this idea of you know just get rid of DNA we
Have other ways of amplifying um DNA which is basically PCR so you can do this assembly first it’s called G Gibson assembly you get the different pieces of DNA put it together form your plasmid and then you just do a round of PCR that amplifies the amount of that DNA it
Basically amplifies the whole plasmid makes several copies of it you can Rec circularize it and then you have enough DNA to put it back inside the bacteria and this actually works really well we published the protocol and it’s a standard method that we’re using and allows us to engineer these laobi really fast
But the only ongoing challenge is that we can’t really extract the plasmid from plantarum because there are a couple a few more cryptic plasmids there and you always get a bit of a mix of DNA in that and that is something that we we’re in the process of
Soling now the next issue was Finding genetic parts that work really well and the first one is particularly getting promoters that are strong and hopefully even inducible that means you can switch it on with like ipg or or uh temp change and so on we looked into the literature
Again and we saw that okay many common uh inducible promoters that have been used in other bacteria don’t seem to work really well in in lactobacilli and the ones that do really work well are peptide inducers that are not very stable within the body and have very limited applicability we have to keep
Delivering them within the body to activate so you want light or temperature or some other thing that’s easier to apply within the body so so we started looking for these kind of promoters we first went to eoli I told you about the thermoresponsive circuit where you have this tlpa um
Repressor that can turn on and off with the changes in temperature um we first went with this because there was suggestion in the paper itself that this could work in lactic veline for different reasons so we tried this out we put this in the uh in the plum strain
And we saw that there I mean there’s some natural temperature responsiveness of the of the promoter itself you have a five-fold difference but the repressor was not doing anything so if you see here the gray bar is with the repressor the black bar is without the repressor
And basically uh the repressor is not having any effect the both of them are pretty much the same but what we did discover was the promoter itself was much stronger than any other promoter that had been described or that had been reported for this bacteria till then so all these
Black bars below are promoters that had been reported in literature before and our promoter was producing five times more uh of the of the protein and so what we did was we engineered the bacteria to produce a fluoresent protein a red floresent protein and you can see
That the ptlp promoter the pellet uh in the image is slightly more colored than the other ones this is already quite exciting to see that we found a promoter just by chance that worked really well hit then we looked at another source okay can the Genome of the um of the strain
Itself provide an interesting promoter and particularly there’s a lot of Regulation gentics whiches around sugar consumption uh around the metabolism of of sugars Within These bacteria so we found one promoter that we could actually engineer in a in a pretty decent way this was a sucros inducible
Promoter so the idea is that it’s a promoter in the strain itself that when you add sucrose it gets activated we took that promoter we put it into a plasmid made it produce this floresent protein what we saw was not really great so with sucrose we could have a little
Bit of Activation so the zero Millar bar that you see here is basically leaky expression and then with sucrose you get a little bit more but what we actually realized was there was a lot of interference to this promoter from other sugars in the media so if you made a
Media without other sugars then you could actually have really good regulation so without these sugars you have pretty much complete suppression of production and with sucrose you have 114 times increase which is great but it’s inconceivable to think that within the body or within most media you would not
Have other sugars so this is the challenge that we are currently in the process of solving trying to eliminate the influence of other sugars in this promoter then we looked at a third source which are bacteria fages which are a really amazing source for genetic
Parts some of the t7 promoter and the t7 polymerase that I talked about in eoli came from a bacterial phas and bacterial phases are just viruses that infect bacteria they need to have genetic parts that are very specific to the host but also to some extent orthogonal to the
Host so they can perform their own functions so what we did is we uh set up a kind of workflow where we identify different bacteria we selected promoter and repressor systems um uh that that had certain properties that we were looking for we designed we built this we
Tested it and we had some methods to improve it well long story short we ended up finding a promoter that worked incredibly well so I talked about ptlp being five times stronger than everything else this promoter was seven times stronger than the ptlp so what you
See this really bright red um pallet on the on the left top left of the few images uh that is the fluoresent protein producing bacteria the pellet of the floresent protein producing bacteria then this promoter had a repressor of that was associated with it in the Fage
And that repressor is able to completely block the expression uh from this promoter and so you see the 475 times repression in the bar right there uh which is basically almost complete blocking and what we’re also excited about is that with this kind of expression level we’re getting
Lactobacillus to the level of expression of eoli protein expression of eoli eoli is kind of the King right now and and we’re kind of trying to bridge the gap over there um so that was something that we were actually quite excited to see that we’re getting quite close the
Ongoing challenge is now we have a promoter we have a repressor we want to make this repressor inducible and there are quite a few interesting protein engineering methods that we’re trying out and we’re starting to get some interesting results out of that all right but with the parts that
We already have at least we have strong promoters now and we have a very easy way of engineering these bacteria so we’ve already started engineering different type of therapeutic compounds for these bacteria to produce and release and so here are some of the examples we have alfin growth alafin
Which is a neutrophil elast or proteas inhibitor nerve growth factor which induces nerve regeneration and some peptides that have anti-inflammatory function so we are able to show that uh whatever proteins are being secreted are active so for example elafin is able to block this proteas neutr elas um the
Nerve growth factor is able to induce uh neite formation in these pc12 cells and the peptides themselves we in the process of studying the anti-inflammatory activities which is a bit more of a complex thing to do but we do know that they’re being secreted because we’ve attached them to a nucleus
And what that nucleus does is once it’s is secreted it can cut up DNA so it can basically cut up DNA and then there is a DNA’s agal essay and what you see down there is basically uh an agar plate where you have a colony of bacteria and
If that bacteria is producing this nucleus the nucleus goes around there’s DNA within the agar that’s fluorescently labeled and the nucleus choos up that DNA and creates this dark Halo so we know that the bacteria is producing and releasing these peptides linked to the nucleus U protein
Enzyme okay and now that we have the bacteria producing all these things we’ve also been able to show that we can encapsulate them in similar hydrogels as I showed before they are able to survive in these hydrogels and able to grow into these hydrogels from single cells into
Confined colonies so you see the bacteria grow into a colony and then they’re kind of limited in the size to which they can grow and we also able to show that we can get things into the hydrogel to activate the bacteria and then whatever the bacteria is producing
Like the nucleus that I just talked about can come out and be active outside and so that’s what you see in the red um images over here when you have no secretion the circle is basically these hydrogels bacal hydrogels that I talked about uh when there’s no secretion
There’s no dark halo around it and when there is secretion of the nucleus a when we activate with this peptide then you do see this this dark Halo forming around so we have the encapsulation we have the production all of this working quite well and now we’re going to test
It with the therapeutic proteins that I talked about now that’s largely the end of the the my talk um what we do do in collaboration with other groups here especially the dynamic biomaterials group here at inm is to encapsulate these bacteria in different formats all the formats I showed you were fine for
The lab but are not great for application in Therapeutics so we pair with them who have really nice uh fabrication techniques like bioprinting microfluidic encapsulation and Electro spinning to make different formats that would be suitable for different organs within the body and we’ve also done quite a bit of
Work to engineer these materials along with that same group uh so that we can control the population size of the bacteria this is something that I said right at the start we are able to control the population size so if you see the the images below the bacteria
Grow into these colonies these little blobs that you see are from a single bacteria within the gel they’ve grown into a colony that you see on the right The Colony has a number of bacteria within and the material ensures that the bacteria Colony size is only that much
But it also keeps the bacteria alive for a long time like for a month or more that I showed before so there was quite a bit of material engineering that went into this we understood certain aspects of how material and bacteria kind of influence each other in these parameters
And yeah you can check out some of this work that we we’ve been publishing in the past and we are still exploring to try to see if we can get materials the materials to actually have properties that improve the performance of the bacteria within them now all of this work has been done
As part of uh myself in the livet science campus um which is a big Consortium that t actually introduced uh and we are able to collaborate with others within the region to to do this work and final shout out as as T said we Host this or we conduct Host this
Engineered living materials conference every year we’re going to have it again this year in September so save the date if you want to see what our conference is about you can see it at the links below um and yeah I hope to see some of
You over there um this time so I’d like to thank everyone in my group that was involved in this work all the funders and all our collaborators who really supported us uh in this research and thanks a lot t for the invitation again and thanks everyone for listening fantastic uh amazing I
Actually have a lot of question but I want to ask p i mean comment on your talk or ask question so should I stop sharing oh you can do it whatever you want to do yeah okay I’ll stop it for now yeah I also have several question so
Um the one is um you know I’m an RNA person have you ever considered to use um rer switches instead of um of uh inducible perod especially in in bacteria there is a set developed by CH Gan years ago with sopine they work pretty good uh in in stepto misis for
Instance and J show it for several other bacteria so I think it’s worth at least trying them I’m not sure if they are already um um yeah optimized for your organism but he he developed a set of six different um um rubber switches and um at least in our bacteria that they
Had a 300 fold dynamic range so it this was really very good and um so simp fun to think about it no but but we have actually so this is something that I am interested about because when we went to lacto basili uh we knew that a lot of transcription factors and whatever
Polymerases these are not going to work so well there promoted from an organism but rival switches do have a better chance of being compatible we tried the 4u thermos switch which did not function too well and I know that there’s been problems there uh and yeah I I think the
Limitations basically are expertise with rival switching so I think so I I think I I have never he said um this r with these tho thermometers that that you can apply them robustly so but um these swiper switches I can send you an public a and and we can also provide
You with this with this um construct so I I think it’s worth trying them we we um they were not developed by us we only applied them but I think they are really cool switches so no but I’m I’m very happy for that suggestion thanks for this connection yeah the other uh
Question I had was this dine or the D is this a huge um cluster or is this only uh are this only a few uh CH so nor it’s not it’s not a huge cluster no so it is the peptide and basically just three other enzymes uh that that
Modified and even secreted from the bacteria it’s not very big so because of these natural compounds they they have huge um chin clusters for for synthesis so yeah I think that’s why there’s a a lot of excitement around darobactin uh because I mean antimicrobial resistance is an issue and finding new antibiotics
Is an issue uh and these huge clusters make it difficult to upscale production in industry and so darobactin is one of these examples where you could do it in Eola you could do it in other organisms and you can try to get higher titer it still is you know going to be more
Expensive than other antibiotics that are actually produced in in quite High quantities and you get pretty cheap which is why we actually looked into having this living therapeutic material approach for delivery to kind of try to reduce the the cost barrier there a little bit are there already known
Resistance against this yeah well I mean bacteria do develop resistance against this um so yeah just like with any antibiotic it can happen but the the incident is apparently much lower um and yeah it’s basically a new antibiotic in the Arsenal that you can use once you kind of exhausted the
Others and maybe one last question from my side um I was always wondering if you have this hydro chells and the bacteria implanted and then you you bring this to um to your to a Target to a wound or whatever MH how is the nutrition Supply or or um how long can
You keep this in the in the organism when is everything wasted and and have you ever thought about this yeah no absolutely I me upake of of met absolutely so our bacterial selection themselves was uh partially based around this so we want we try to see which bacteria grow naturally in an organ
Where you want to get the therapeutic there so that means those bacteria naturally have the nutrients that are needed to survive so a lot of the results that I showed uh we actually grow these living therapeutic materials or we grow the bacteria with mem and cell media so the media that are
Suitable for human cells basically um and I know the the the nutrient availability in different organs are different so we have some projects where we’re we’re trying to have this delivery in the eye like on the ey surface and there we go for a Cory bacterium strain that’s naturally found in the conjuctiva
So I didn’t talk about it this time but the the the focus is to get the bacteria that naturally live there and naturally survive or thrive on the uh nutrients that are available in that part of the body um but yeah how well that translates into drug production is
Something that we need to find out with indivo studies that you know we’re getting to in pretty soon Patrice do you have any further question not yet thank you okay so I I’ll ask a lot of questions so the related to her last question uh so that’s very important once you
Successfully demonstrate that happened that’s fantastic uh but related question to that is uh basically when you think about real application onite you know human body cut you know skin whatever you want to do the experimental condition in the lab very well mixed condition sometime even if you use the mimicking whatever system
Still you kind of use the well mixed system but that’s not necessarily true for the gut skin you know human body and then I guess I mean have you you know what is your perspective or Insight regarding that Point that’s my first question yeah I mean we we’ve looked
Into this in different organs and then different organs do offer different kind of nutrient environments so one of the more challenging ones is the eye surface because all you got is tear there which is very limited in terms of nutrients um but that’s why we take the the bacteria
That grow there naturally um how that’s going to work out and and translate into drug production is something like we really need to see in in Vivo studies but what we what we like we we gain confidence from the living Therapeutics clinical trials that people have been
Doing you know they’ve been doing it with lactoval with eoli not just only in the gut but also on wounds and and within the blood uh for some cancer studies and so on so in in those cases we do see that these bacteria do thrive in the locations where they’re supposed
To be affective sure and and yeah that’s that’s kind of the the the motivation uh you know the to to select certain types of bacteria from those studies and those reports uh and eventually you know my my vision would be uh to not just take these standard strains but how you do with
Like car T Cell Therapy you take it from the patient you take a strain from the patient that works really well for them engineer that and put that back in sure and you know to have the best kind of performance in there I mean that’s a
Long way off to to get there like already working with the strain you’re not used to every day is a challenge but I feel synthetic biology in general is moving towards being able to do these kind of things okay I mean thank you for defending for me as well because I’m working
On a lot so my second question is uh more question or more comment but the your light in this sub system time in the four part of your talk is amazing I see a lot of potential light uh I guess that’s probably more useful for skin
Application than gut because to me if I remember correctly blue light not penetrating into the gut right yeah yeah absolutely so blue light uh blue light is not ideal there for sure um ideal would be infrared light but even then you get only a little bit of
Penetration within the body um there are some technologies so there are now the same person who made the blue light inducible system Andreas Mish from bid in Germany uh he’s also made red light inducible infrared light inducible systems very recently so these are things that we can adapt and get to a
Higher Wavin Spectrum but there’s other work in the Institute itself where people are developing these kind of hydrogel optical wave guides M that can transmit light from outside to within the body and these are not like glass based optic wave guides are much more suitable for our tissues and so on got
So the idea is to even in these hydrogel wave guides to encapsulate our bacteria make that into a living therapeutic material and have the delivery wherever it’s needed within the body of course that’s that’s a surgical procedure but uh if if necessary that would also be
Possible I see but it’s also a reason why we move to the temperature responsible systems also definitely so the the red red light probably not going to penetrate much through the skin right about about a millimeter infrared is about a millimeter you may consider the ultrasound there are cool papers
Multiple paper from kch you know saap yeah yeah exactly I mean that’s that’s the same Thermo induc system that we have have used and the idea would be to use focused ultrasound ex we have actually another paper where well again it goes back to light but where we use
Uh we did in collaboration with another group here uh we use Nano rods within our living therapeutic material to convert infrared light more efficiently into heat so in such a way that if you apply infrared light you can apply it at a dosage that doesn’t heat up the rest
Of your tissues rather just the material inside yeah exactly but again it’s infrared and then another kick question because I want to mind for for about time another key question is you you show the one cycle of light or no light you know did you test multiple cycles of
That one yeah we we did test multiple Cycles but eventually for the publication we didn’t do too many repetitions of that I mean it it is again it is it’s this format with the glass thing at the bottom so it wasn’t the ideal format that we were going for
So we didn’t just push working there we wanted to move towards more suitable things so we we the proof of principle was enough for us right now but yes you can make it on and off multiple times got it got it and then it’s more likely
Common uh you know we l to publish one paper regarding antibiotic rissant gen free groning system in report I believe method so you you know I happy to collaborate with you uh implementing that into your lactobacilus you know strain thing because we we now need to expand the tool kit you know another
Probiotic as well so you you did it in Nile I guess right yeah we did but we want to expand that to other BAC okay yeah i’ I’d be very excited anything that is EXP int yeah and then the last question is uh also a bit common but to clarify your lacto
Basus uh stancing that didn’t work but with the increasing temperature the signal increase in somehow yeah and then I probably missed the why you may exp I didn’t I didn’t really explain it so the we don’t know exactly or we have an idea of the sigma Factor that’s actually involved in driving transcription
Through that promoter so we tried many parts I just showed one we we tried many parts we didn’t expect promoters to just naturally work when we took it from eoli or so on that promoter was particularly taken from Sal Monella and engineered into eoli by Mel Shapiro uh and so again
We didn’t really expect it to work we put it in there and somehow the sigma factor is active and the the only thermal responsiveness is because that Sigma Factor itself seems to be thermoresponsive and the thermoresponsiveness is like from 30° to 39 it’s a fivefold increase so it’s you
Know I don’t know how much uh yeah yeah if it’s a overall metabolic thing that that Sigma Factor itself is up regulated or down regulated or if the sigma Factor itself is thermoresponsive but you know what among all the data other data is amazing but I love that data the most because
No no because that’s not successful data but that is a very important lesson MH yeah showing to all the young people the importance of control you do not include the control you know without you know repressor you you never know you may think that’s working but it’s not so oh yeah tell
Everybody you know young people especially how important control is and then and then please include the control that’s my last point on to make and we had we had that wow moment for for a day before we included the control then that is the end of that
Okay so I I probably went to probably W it up because I already see the 8 minute after o clock so let me Closs and then we may chat more uh with some audience okay so thank you all for joining and staying today uh in this
Special day I keep saying that uh we’ll meet again on March 7 Thursday the same time the same June link we’ll have Professor Jing Wang at North Eastern University Professor Kyle laurenson at King abdalah University of Science and Technology cast uh another in the midle East uh country uh
Expert uh as usual the followup informal chat will occur without recording please stay here if you’re interested chatting with us uh I will promote you to panelist who can speak and show your handsome and pretty faces if you wish thanks and I stop recording thank you just give me one second