Introduce Chris Chris did his bachelors at Michigan in chemical engineering and from there he moved to Caltech to do his PhD and Initially I was confused because I saw three names as his PhD advisors thought perhaps He just had bad luck and move from lab to lab to lab, but he tells me now He took three P, you know professors to mentor him simultaneously at Caltech and He became a biochemist by a physicist there from there. He moved to Berkeley To work with Adam Arken After which he started his own position in 2003 so roughly ten years ago He moved to MIT about two and a half years ago, but while he was at UCSF He did some really beautiful studies that actually define what we think of synthetic biology today He's one of the pioneers the leaders of the field and for that He won a bunch of awards the Sloan Fellowship the Pew Fellowship the Packard Fellowship the NSF career award and it goes on I'm just highlighting a few before I bore you to death and the indications were there early on He had one of the most prestigious post-doctoral fellowships and the NSF pre-doctoral fellowships and To top it all off He won the MIT's famed TR the Tech Review Awards in 2006 to be one of the few Innovative leaders at TR 35. I believe is what it's called He's developed a variety of tools that are now being used widely across The field and is now also the chief editor of a newly launched journal the American Chemical Society Synthetic Biology and Some of the you know beautiful work that's being done is now appearing in that journal so it's already setting He's bringing his vision to fruition and engaging a larger community. It's a pleasure to have you here Chris Well, thank you a seam. It's always great to be here. I always really enjoy visiting Wisconsin. It's you know fantastic nexus of Chemical engineering and the engineering sciences with with microbiology and of course biochemistry and so on so it's always nice to see What's going on? So I'm coming from the background of engineering my background is chemical engineering and I say that because this is a biochemistry department And so I want to make the point that our primary interest is being able to build systems that don't yet exist and To push the scale and the sophistication by which we can do genetic engineering And so I'm going to start out in this talk just by giving you a little bit of an overview of the types of problems That we hope to be able to address in the near future and then I'm going to walk through more carefully a story Where we've been working to simplify some of the genetics around nitrogen fixation Which is a function that has been tried to be transferred between organisms for some time and Try to leave you with a sense that there is a phase change that's happening now in the size and Scale of designs that we can do in genetic engineering So for a long time We and others in the field have Drumped of being able to program cells to do complex functions. And so these are are some examples of either what has been envisioned of what could be done or Things that happen naturally in biology and so for example In the example on the left This was a student of mine postdoc mine who then went and joined UC Berkeley Chris Anderson Where his whole dream has been able to engineer bacteria as therapeutic devices Where they go through different micro environments in the body they sense where they are they perform a therapeutic response in each one of those Neaches and all of this would be programmed in the DNA on The right I'm showing a number of different materials that are naturally produced So cells are the natural architects of the nano world I'm showing a glass sponge, which is essentially spinning fiber optic Cables as well as diatoms that live in the ocean and build these very intricate silica structures So everything on this slide involves being able to program a cell to go through a complex series of tasks And it's a huge difference from what is currently being done in biotechnology so if you look at the forefront of companies like Dupont or Dow ag or even startups like Refactored materials or amorous they're really about the production of individual small molecules and really just having biology produce that But that's not getting close to what biology has the capability of doing and so everything that we're trying to do is to Bring that scale up where we could actually be able to undo these types of functions in cells and very broadly There are two things that are required in order to do this The first is that you have to be able to manipulate many genes possibly hundreds of genes And you need to be able to control precisely When they're turned on and under what conditions so the dynamics and the conditions and so for that you need synthetic regulation and so the way that we envision this all coming together and moving towards Being able to do this is on the scale of whole genome design Where you could actually go into an organism and be able to construct systems that are on the same scale as the natural genome and We very roughly divide this up into different categories Where on the far right we have the actuators or these are the things that the cell is actually doing These are connected to circuitry that's telling when the different systems to turn on Which are then subsequently connected to sensors which are looking at the individual environment and Dictating when things should be activated so for example in this Fantastical idea of creating a cell that can spin its own antimicrobial cloth You might have a set of genes that are there encoding silk proteins You have another whole set of genes that are making antibiotic nano particles The silk has to be exported at a certain time and then modified and all of this has to be integrated into a large system and so My lab is focused on each one of these steps So being able to sense Environments to be able to process that information with circuitry and then to control different outputs What I'm going to focus on in this talk is the last of those steps So being able to convert any cellular function into an actuator that can be turned on and off whenever we want and to be able To transfer those cellular functions between organisms as easily as possible So now we've been doing this and others have been doing it for some time and this has really been facilitated by the advances in DNA sequencing and DNA synthesis so over the last decade to two or three decades now of sequencing There's been sequence databases that are populated with genomic information. That's encoding functions and all of that information is Stored in inaccessible unless you're able to go in and re-synthesize the DNA that's encoding those functions And this was facilitated about a decade ago with the rise of DNA synthesis companies that allow you to go and imprint out A lot of pieces of DNA or a lot of genes and screen those for a particular function And so this has led to an area done by a number of different labs called part mining And this is where you have an enzyme that you think has an interesting function But instead of working with that one enzyme you go in the databases and you print every single Enzyme that has that potential function and screen it for activity and so this is One example of that where we were looking for an enzyme that produced a methyl halide for sort of random reasons But instead of working with one gene from one organism We printed 90 enzymes from 90 different organisms that had any chance of performing this function And then we identified the top ones which came from plants and from Bacteria that hadn't even been isolated just part of metagenomic samples and so on So this is a very simple single gene experiment where you're transferring this function from a plant or from a microbe Into a new host in order to take on this new function And the reason that this was possible of course is all of the tools that already exist in genetic engineering So we had to use synthesis to access the gene But then we just pop it into an expression plasmid that has a inducible promoter and a ribosome binding site So we can turn the gene on and the new host and so on So it's a very simple type of operation that allows us to access that function and then turn it on in a new host So then we started to get interested in functions that can't just be encoded by a single gene But require many genes collectively activate turned on in order to to produce that function and so this is where we started to get interested in gene clusters and so what happens in bacteria is that many cellular functions have all of their associated genes encoded in a contiguous region of the genome known as a gene cluster and so for example one of the the system I'm going to talk the most about today is encodes nitrogenase activity and this is a 25,000 base pair region of the klebsiella genome that has all of the necessary genes for this function as well as their regulation and there are gene clusters that encoded just about anything that you can imagine a bacterium doing so for example We also work with a protein secretion device out of salmonella, which is 35,000 base pairs of DNA And again that has all of the necessary genes to build this secretion device and export proteins from the cell There are many many examples that produce pharmaceutical like compounds Things involved in the breakdown of biomass and energetic harvesting Including things like light and so on and so in this this vision of the future What we would like to be able to do is to look out at the microbial world and take any willy-nilly functions from different bacteria that we want and plug them together into this single organism that then has some combination of Abilities so for example if I want to be able to export protein I want to be able to pop in that unit of DNA that Encodes the protein secretion device if I wanted to fix nitrogen I want to pop in that piece of DNA to fix nitrogen and so on So now the challenge with it is that instead of an individual gene that we could just put in front of an inducible promoter You now have 16 or 20 or 40 genes that all have to be turned on at just the right levels in order to get activity And so this is just a subset but with all of the really high throughput sequencing that's been going on There are literally tens of thousands of gene clusters that encode just about anything that you can imagine and this is being Found in a very automated way the bioinformatics exists it already go in and I Identify these different types of functions and it's growing and scaling with the size of the database And so when we look it we often just see all these different functions So we want to be able to move from one organism to the next and so there are really three things that you need in order to Do this transfer The first is that you need sequencing and bioinformatics and that's so that you have the Information associated with that function and you have some way of going into the databases and being able to figure out What is the subset of DNA that is required for that activity? But that sequence information is not enough to to obtain that function You also need DNA synthesis and this is because you need some way of being able to re-access The information and turn it back into physical DNA, but you can't literally re-synthesize the DNA sequence from the organism from which you obtain this because all of the Regulation is going to be different between that and the new host So the third thing that you need is Synthetic biology and this is what allows you to then replace the regulation of that gene cluster so that you can get it to function in the in the target organism So we started thinking about these different things and moving from individual genes to sets of genes and and trying to be systematic about it and so one of the first problems that we decided to start trying to address is Is nitrogen fixation? And this is a problem that's been articulated for 30 years or more Where the problem is that? When we look at organisms when we look at plants and their ability to obtain nitrogen from the environment most of the Plants that we consume are not able to obtain their own nitrogen so things like legumes and beans are able to form associations with microorganisms that deliver Nitrogen, but cereals like rice and corn and we are not able to do this And so the way that we've solved this problem in agriculture is through the chemical production of ammonia Through Haber-Bosch and usually through the burning of natural gas And so in this way the cereals are consuming the nitrogen out of the soil Which is then being put back in there through chemical routes And this is what has led to the incredible increases in in cereal yields over the last decades and so since the 1970s it's been viewed as that as an alternative to this chemical route of Synthesizing ammonia and dumping it onto the soil that what you'd really like to do is to take one of the Plants that is able to associate With these organisms in the soil and transfer that capability from a legume to a to a cereal crop through genetic engineering and this was articulated at the same time that the recombinant production of insulin Was articulated in the 70s at the asylum our conference that quickly was solved and it became Genentech and I think the reason that was solved quickly is because at the end of the day It's a transfer of a single gene into a new organism Whereas as you'll see for nitrate nitrogen fixation to require many genes for activity They have to be balanced just right and the tools haven't been there in order to actually achieve that And so we decided to start going in and try to figure out what's stopped This ability to transfer this function between organisms and then how the tools of synthetic biology might be able to help with that step So this is the gene cluster that is the model system for nitrogenase activity. It's from the Bacterium klebsiella. It's a 20 gene system that's encoded in 25,000 base pairs of DNA and it has the core genes for the nitrogenase itself Which is what performs the conversion of atmospheric nitrogen into ammonia It then has a series of genes that create the Fimo co co-factor and then loads it into the nitrogenase enzyme It has additional enzymes are involved in the delivery of Electrons to the reaction center and then it's got a regulatory network that very carefully controls expression and so one of the first things that we did is we went into this system and We wanted to look at how sensitive it is to changes in the expression levels of the individual component Proteins, and so we performed a series of experiments like the ones I'm showing here where we would go into the native gene cluster and knock out either an Individual gene or a small set of genes which we then would complement back onto a plasmid So that we could sweep those genes through expression levels and look at the impact on the activity of the system And what we found is that this is a very fragile system where if you look at it funny You very quickly lose activity and so you can see that Here where you have to balance the expression level of the genes just right and if you're too low or too high You very quickly lose activity and each of the of the subsets of individual genes and combinations of genes have this property And it's not that they all have to be high or low Each set of genes has its own optimum level that has to be just right in order to get the activity of the system as a whole So now what I'm showing with these six graphs is just one slice through this space Where we're changing the expression level of a single gene as soon as you start to do combinations of genes it gets much more complicated So for example, if you're looking at the expression level of two genes in this system Each of which has an optimum then you create this sombrero hat like space Whereas you change each of those genes from the optimum levels you very quickly lose lose activity So now in a system where you have 16 genes all of which are very fragile and have to be carefully optimized You then don't have a two-dimensional space. You have a 16-dimensional space Which is this hard to imagine volume wherein somewhere in this space You've got a volume that represents all of the combinations of expression levels that are consistent with activity And if you're outside that combination of expression levels, you then have an inactive inactive system So I know it's hard to think about 16-dimensional spaces and when I was a student at the University of Michigan My advisor was Richard Goldstein and he used to tell these really bad math jokes And one of his math jokes is why should you never buy a hyper-dimensional water melon? And as it turns out as you increase the dimensionality of a space the surface area over the volume goes to infinity And so as you increase the dimensionality most of the surface most of the volume is on the surface And so the reason you don't buy a hyper-dimensional water melon is that it's all rind and That's bad. I warned you the So what that means is that when you're in this space You're always on the on the edifice of falling off and losing function. And so in a transfer experiment Where you're taking a gene cluster with 16 genes and you're cutting out and you're popping it into a new organism you're even if everything works just right and You know all the ribosome binding sites and all the promoters and all the terminators and all the codon usage And everything is just perfect. It's not going to get the expression level just right And so if you wiggle all those expression levels you end up moving into a new space and losing activity very quickly okay, and so then you have to try to regain activity, but because the The system is so redundant and so overlapping and and so non-modular It's impossible to make these substitutions. And so you're never able to regain activity And so we've articulated the problem like that and then there are certain roles that synthetic biology can help with this process So on one thing the way that we think about genetics is very different And so I'll show you how we can go in and simplify the genetics of a system to make it more conducive more understandable for transfer We also have tools to allow us to very precisely control expression levels So that after the transfer you can then go back in and tune the expression levels to be just right to get activity Some of the part design that we've done for E. Coli and other organisms can be applied to new hosts and then I by the end you'll see how our ability to construct very large and Sophisticated pieces of DNA actually helps us with this process tremendously So first I just want to go over some of the some of the aspects of a gene cluster that actually stop you from being able to transfer it and So this is what I call the nice nature reviews microbiology view of Genetics and so when you look up a particular gene cluster, it's all very nicely organized But the actual genetics underlying that can get very very complex and it's that complexity that becomes a problem So for example, there's some internal regulation But then the system is going to be embedded in the native Regulation of its of its wild type host and this is often in ways that are that are poorly characterized The system also has very non modular and overlapping part functions. So for example You know over half the genes in the nitrogenase gene cluster are overlapping So that the ribosome binding site for the downstream gene is in the middle of the upstream gene And that's just a common way that you have systems are encoded But now if you want to try to change that ribosome binding site There's no way that you could do it without interfering with the coding sequence of the upstream gene So that's a very much non modular system. That's interfering with being able to make Substitutions in order to achieve an engineering goal And there are all types of examples of a genes encoded within genes even operands are a problem and so on There's also a very complex Encoding where you have a lot of overlapping functions and you often see this within open reading frames And so this is an example of a review paper that was looking at all the different ways that you have promoters in in a In a gene cluster and so you've got a lot of examples of promoters within genes pointing upstream You have promoters going against the tide of genes for no reason promoters pointing at no target and so on and So you have all of these overlapping functions and this stops you from making certain types of moves as you're trying to build a genetic system So for example, if you took this this gene here with promoters going the wrong way And for whatever reason if you wanted to move it to the front of the cluster and you found that that was a non-functional system It may not be the move itself That's the problem, but the promoters pointing the wrong way that then turn on something that you didn't expect that then causes the problem And then these are just all the things that you know Of course, there are all the unknowns of biology and even the most well characterized systems that are constantly you're peeling the onion Trying to understand more about how the genetics are encoded So as one example, we were trying to move a promoter from one organism to the next and it killed our target And we couldn't figure out why and we gave up and then years later There was a paper that showed that there was a small RNA encoded there that interacted with porins So that when you tried to move that piece of DNA it then killed the cell because it it interfered with the porins And so you've got a lot of examples like that that interfere with your your ability to to move things around And so we had to come up with a way that very systematically Fixed the natural system to get rid of all of these issues Simultaneously and so to do this we turn to some of the the fundamental tenets of synthetic biology So at the core level in the field we think of Genetic parts and so these are units of DNA that have some biochemical function like a promoter We're a ribosome binding site or an open reading frame and so on and we tend to associate a single Genetic part with a single function and that's already quite different than the natural genetic systems where you have these overlapping parts Where you might have multiple functions within one one part So then based on those parts you assemble them in order to create a genetic device And so this has some human identifiable function It might be a pathway or a circuit or a sensor or whatever That then gets assembled into a system That's a unit of DNA that you then put into a cell that performs that function And so we've got this hierarchy from parts to devices to the system as a whole that then goes into an organism Now we wanted to apply this to the problem of gene clusters by flipping it on its head and so We created this process called refactoring which tries to go in and Systematically reduce the system into its component parts and the the term is stolen from the software industry where If you have a piece of software that's crashing like Microsoft Word or Windows One thing that you would do as a software engineer is go in and refactor the code and that means you just start from scratch You completely rewrite the code to create the same program But where you've organized the code to be modular and engineerable and stable and so on So the way that we're applying it here is where we're going in and completely Rewriting the DNA sequence of the gene cluster So that it's modular and engineerable and so on but that it encodes the same function as the wild type system And so what we do and we mostly do this on the computer is we go in and we have have a gene cluster and We start out by getting rid of all of the non-coding DNA so anything that's not an open reading frame gets thrown out We then take the open reading frames And we go through a process that we call code on randomization And this is where we try to select codons that encode the same amino acid sequence But produce a DNA sequence that's as far away as possible from the wild type sequence And the idea there is that we create open reading frames They have so many mutations in them that they eliminate all the internal promoters and small RNAs and Operator sites and so on and so forth Just by random chance. And so we have genes that are inert for everything other than Encoding the amino acid sequence. They're supposed to encode and at the same time that we're doing the code on randomization We're also running all of these algorithms that we have to find these functions and we eliminate them when we do find them We then organize the genes into artificial operons So these are organizations that make sense to us, but not naturally not necessarily the native organization We then add synthetic genetic parts that we've characterized in isolation For example ribosome binding sites and terminators and so on We then use a phage polymerase promoters that control the level of transcription and Then the last step is that we create what we call a controller. And so this is the usually a genetically distinct Construct that we build that has the synthetic sensors and circuitry to control the timing and the conditions for expression and then this the output of the circuits Then produce the RNA polymerases which then go on and turn on the pathway So at the end of this process, we're left with a gene cluster that has absolutely no DNA Identity with the wild type. It's been designed to be modular and organized We've gotten rid of all of the native regulation and replaced it with this controller that when we pop in the cell it then is encodes when and The dynamics of expression and the remarkable thing about this process is it works And we we published the first version of it last year so this is the example of the refactored nitrogenase gene cluster and It's a little hard to look at at first But as engineers this picture what makes a lot more sense to us than the nature reviews microbiology paper and the reason for that is if you look first of all It's it's highly modular So each box is a unit of DNA. That's a part that has a prescribed function Where we know what that function is doing because we put it there And we've independently measured it. So for example if we look at it's very hard to see But if we look at each of the ribosome binding sites in a system You'll see a number underneath them and that's the strength or the expression level that that ribosome binding sites achieving And you see the same thing with promoters. We know the strength of that promoter Terminators even we know the strength of that terminator And so each one of these these parts has been put in the in the system for a purpose And then we can control the the expression of the entire 16 gene system with a separate controller Which encodes this logic function? And so here we just have two small molecules that are that are inputs and we can turn on the entire 16 gene system When those inputs are in the right conditions of that logic, and then it stays off otherwise And so we've gone in we've made this modular system We can drop in a controller control when the conditions that it turns on and so on And so we've have this improved activity or this improved Reduce the function it actually comes at a cost of activity. So the process of simplification Initially created a system that was much less functional than the wild type system and you can see that here Where in the in the wild type we have a high level of Nitrogenase activity and then in the first version of the refactored system. We only recover about seven percent of Activity and so this this process of simplification came at this cost and so we started to look back and The the process of going through this actually took about seven years to create the first Refactored system and so we started to look at well, why did why was it so slow? What were the things that allowed us to speed up during that time so that we might get faster in moving forward? And so I just have summarized those seven years in this one step, which is the first Subsystem that we tried to work with in this pathway where we started out by trying to refactor one of the one of the Operons in the system that only encoded five genes. This was back in 2004 And what would happen is that on the computer? We'd create a refactored version Send it off for DNA synthesis. It would cost ten thousand dollars to synthesize this thing. This was a while ago And we just completely lose activity so you get no activity And then just to give you a sense of the scale of the of how quickly we were able to fix things We would then have to go in and we could build about five at a time Or we would cross the wild type in the synthetic trying to figure out Exactly what part of the refactor system was causing the problem? We would then figure out Okay, so the the first nif H gene that we tried to Code on randomized created in this case transpose on insertion sites which then destroyed the activity of that gene So we'd have to go back re-synthesize the gene Find that it worked but not at an optimal level then manually try to Tune the arrive his own binding site until you recovered activity. So in this sort of design-built test cycle We were doing about five at a time with a very small region of the cluster and trying to identify Improvements and then we would have to build the entire cluster and the tools during this period were just not there for this Where we would have to assemble about a hundred or so genetic parts each time So we would do it in a hierarchical assembly So what I'm showing here are all the different portions of the cluster along with the percent of activity And so with this method Gibson's assembly came online at the tail end, which actually is what allowed us to build these at all So we could assemble them into these larger pieces of DNA But we lose activity very fast and so this half clusters at ten percent of activity Which is the multiple of the sub clusters and then here we'd get three percent which again is the multiple and actually the first system that we assembled Took like four months and we only got the point three percent activity We're actually really excited by it But you had to hold the paper just right in order to see any activity So we would go back you'd have to go all the way back to the beginning Reoptimize those that were the pinch points Rego through this process of assembly, which would take a long time and we got one that was functional And that's what we published as the first step, but we said after seven years We've got to do something differently right we have to approach this problem in a fundamentally different way And so we started thinking about well What's actually the ways that we're going to do this faster and there are two general ways that you can think about optimizing this process The first if you have a design build test cycle where you're designing a construct You build the physical DNA and then test it and then learn from it You could imagine going faster by going through each of these steps faster So you go through more design build test cycles per unit time We decided to do a slightly different approach And this is where we partnered with the Broad Institute to take out massively parallel approach Where instead of designing a single construct we would design hundreds of constructs simultaneously Build each one of the those constructs test each one for activity and then apply learning algorithms to go back to the design And so then you could in this very massive way try out a lot of designs simultaneously And so this is a collaboration with the Broad Institute, which is which was absolutely critical in this Because what they did is is they applied manufacturing principles to the process of DNA sequencing and this is what led to all the advances in in It was was a major contributor to The advances that led to the human genome effort and all the metagenomics that's going on so on and This is their facility. It was designed by Toyota and it's really a manufacturing pipeline. It's not an academic lab It's in an old warehouse. They used to store the beer and the popcorn for their Red Sox And so it they're now able to sequence about 4.3 trillion base pairs per day Which is absolutely incredible So we decided to get involved with them and retool these processes so that we could do DNA synthesis and assembly at a much higher scale than what we can do now and this is where we We built this thing that we call the MIT Brode foundry and we went in and have been working on each one of the steps One is rethinking genetic design so that instead of building single constructs We can build many and when it designed many in one one in an equal amount of time then build all of those constructs to specification very rapidly test out a lot of of Constructs as well as genome scale transcriptomics and proteomics and so on and then learn from that enormous data set that you get And so we set this up as a pipeline they they're manufacturing people went in and have have started Working out all of the limbs and how you how you get the computer aided design to communicate with the construction and quality checking and so on So I'm going to go through some of the academic questions that we had to address So one of the first ones is that we realized that really for the last 30 years We've been designing genetic constructs in exactly the same way and these are tools like vector NTI I'm sure everybody in the room has used one at some point where we think of a genetic design as a plasmid And so if you have a small plasmid with three genes and a dozen parts This is a visualization that makes sense but as soon as you build a try to design a very large system like this is one of our Nitrogen this is the refactored Nitrogenase cluster in vector NTI. It just becomes a total mess First of all just going in and trying to make sure which gene goes where is it is a huge problem You make mistakes There's there's really no way that you would do this 10,000 times, right? It would it would take up a huge effort or you just have to resort to some random method So what we did is we partnered with Doug Densmore at Boston University and Doug had caught my attention because he developed a Different way of articulating a genetic design. It was fundamentally different than How we had been thinking of it and it's Part of what he calls the Eugene language. So instead of that vector There's a code here and don't worry about what it looks like and what you do is instead of Having a single construct that you're trying to build You identify the parts that are in your system. So underneath this file You would get all of the DNA sequences for the parts in the system And then instead of saying this particular construct is this combination of parts You create rules in your system and the rules are anything That you can as an experimentalist write down about how you would describe the genetics of your system So this promoter has this particular RBS all the genes have to point the same way These three genes have to be an opera on that one has to be in its own cistron So and so forth anything that you you can articulate can be captured in this language Written very simply in Eugene, which then gets converted into a machine readable code And this is critical because as soon as the machine can read it the machine can design it And so you can convert this into a into a genetic construct That captures all of the parts and all of the rules between those parts So if you're not used used to thinking about this by the way This is going in vector NTI and so this is this isn't just sort of the fantasy of of our lab But this is I think is a really a direction where genetic design is going If you're not used to thinking about parts and rules between parts we can turn to Shakespeare and Think about how we could assemble words into sentences rather than genetic parts into constructs and So what the simplest way you could do it is where you just have a list of English words And you randomly combine them so they create sentences like iron clothes put adhering behind study But then you can apply grammatical rules on top of that and start to generate Random sentences, but ones that adhere to the grammatical rules like the wife anchors the monkey and Then you can start to apply constraints on top of that system like two oyes before B You can start generating new sentences and learning from those sentences and and so on to create new constraints Which further condense your system and so the way that this works for the genetic designs Is that we can start out by defining all of the parts in a system? And then once we do that we can write a little program that then permutes those parts to come together randomly and as you would Expect you then end up with Nonsensical designs so you have genes without any transcription or translation control multiple RBS is in series promoters pointing against genes and so on so you just get random nonsense But then you can articulate things like expression rules so every gene needs an RBS every RBS gene pair requires a promoter and a terminator and so on so forth You can articulate those as rules and then rerun the process and it condenses that space So now you only create constructs that are at least consistent with transcription translation rules As he's every gene has an RBS There's always a promoter somewhere upstream in a term terminator downstream And then you can add whatever rules you want to apply to your system So for example Have it where three genes are in one operon and then one gene is by itself in its own Cystron and even there in this simple four gene system There's still a huge amount of diversity that's possible with that system And this is one of the real surprises that come that that came out of this where it's really hard to constrain your system to just one construct because all of all of the ways that you can Fluke the system and you can even think of this in terms of if you're building a single plasmid where you're just trying to express a protein You know, there are all these design choices you make that you don't mean like you could Use this multiple cloning site versus this one or flip the whole cassette this way or that way and if you enumerate all those different ways you actually have a lot of Diversity that's that's possible and and you're making design considerations that aren't really there So so the output of this program are these different Constructs, but then this feeds doesn't just make pretty pictures. It feeds into the pipeline for the robotics and assembly process to actually build each one of those constructs and So we use a series of hierarchical assembly methods that start out with small parts that assemble them into essentially individual genes and then and then final constructs and so we started out just by doing very simple assemblies where we were very constrained by the the the system Where we would just do things like switch promoters and RBS is and so on because we had this feeling that the architecture had to be relatively fixed compared to wild type and we would assemble the the The genes for this so this is one example and you can go in and screen this and then learn new new rules from it This is an example of another early library that we built where we added a little bit more diversity but still keeping the overall architecture more or less the same and Again reassembling and then you can see in this case now remember this is not random Library each one of these has the same amount of design information Before you run the experiment, but then after you run the experiment you learn something that can then be rearticulated as a rule back into the design process and so in this case we could figure out a rule Which then allowed us to optimize this Subcluster so we went from less than 20% activity recovering full activity for that system So then we went in and we had started noticing with different projects in the lab that actually The the less we felt constrained to the wild type genetics the better off we were in our library And so we we decided to build this library for one of the 16 sub portions of the nitrogenase cluster that was causing us a lot of trouble and so we built a library that we called the state a library because The state of building is a is a building on campus by Geary that just looks messed up in our library looks messed up so if you look carefully if it in this case Before we were putting all of these constraints like operon occupancy and stuff like that and In this case we just said if we don't know for 100% that it's not a rule We'll leave it out and we'll only put things in the system that we know our rules And we'll just permute around everything else. We'll let everything else float And so we created this library that is one operon in a wild type system But it was up to seven promoters genes pointing every which direction Operon occupancy in order changing Just going crazy. There's a bunch of constructs that get designed And then there this has to turn into an assembly map that takes small pieces of DNA And then combines them into transcription units into build bigger pieces of DNA That they get physically combined and this is all done in parallel And a lot of the software is trying to figure out the optimal way to get the final set of Units of DNA with the smallest number of combinations of parts they have to do So we then screen this particular library And we found that the top hit was really surprising in that it looks absolutely nothing like the wild type system The the wild type system and the first Refactored are both single operons with six genes and our best Cluster had seven different promoters genes changing orientations Just an enormous amount of diversity It's not that that had to be a way to solve the problem We also found one that looked very much like wild type with just different parts in the location And so we found these very genetically distinct Constructs that are coming out of the hits and we can be very quantitative about it I won't really go into it, but we can this is showing a map of each construct where lines indicate similar constructs And just showing that we if we map out the space we see high activity shown in green Spread out throughout the region And we can be very quantitative and so on so when we went in We then had an improved cluster where everything shown in in red is is an improvement And you can see this divided up where the HDK portion got up to a hundred percent the BF Bqf us vwzm is up to a hundred percent in the last bit of in E&J was a bit of an issue So then we once we built this this infrastructure We could then start building extremely large libraries of of clusters again all by design And so this is the example of about a hundred twenty sixteen gene clusters Where we were very systematic about how we varied the components of the cluster and this is about three point five Megabases of synthesis and assembly so it's as though we built a bacterial genome now in the scale of the experiment that we did And we do it at very high efficiency with only four percent error rate meaning that 96% of that library perfect and then four percent have mutations and that's allowed us to then go in and and improve this system further and and All the components are very high and then the top cluster all together is about 60 percent Again showing in red those parts that had to get substituted for activity So what we're finding is that one of the the big questions when we started Was whether when we take this very dramatic process where we wipe out all of the native regulation and replace it with synthetic regulation Whether there was something about the wild type that we were missing about the with the system that we were just eliminating It could never get back and what we're finding is that we can do just as well We're almost just as well with a completely synthetic system, and I think we're very close to fully recovering the activity So now we've started some species transfer experiments and And this is something we're just now starting So one of the simple examples is E. Coli. This is this has been done by other labs with a native system It's just a simple example Looking at how systems change and kind of laying out our strategy for it So we start out with Klebsiella with a system that is non modular And then we refactor it we lose activity. We optimize it to boost it up We can then transfer it into E. Coli, and we lose it back down again And so we see this loss again in activity, but then because we have a modular system We can do something that we couldn't do with the wild type cluster, which is to go in and optimize it And so this is an example where we then built a library to re-optimize activity where we took a numerical optimization approach where we switched between low and high ribosome binding sites and then In systematically learned from that information to drive the next library and so on and so this is where across all 16 genes We're swapping the ribosome binding sites systematically and you wouldn't be able to do that For example, if your genes have their ribosome binding sites in the middle of an upstream gene and so on and so with this We're able to recover activity. So we're starting to take a similar approach looking at more sophisticated organisms that would be the targets of the transfer So now I talked about Nitrogen fixation. I'll just Conclude with some of the other systems that we've gone through this process And I started out with this goal of trying to integrate across the difference synthetic systems that we're working with to build up towards Whole genome design and you can start to see how this how this comes together So for example with metabolic pathways, we've gone in and refactored individual pathways that in this case Just have metabolic products that change the color of the cells and then in the in the controller. We have a logic operation With synthetic circuits that switches between two different phage polymerases that turn on the two different pathways under different Conditions specified by the sensors. We have a nitrogen a simp system that I showed and we've done the same thing for type 3 secretion in Salmonella using a different polymerase So now you can imagine taking these two different cellular functions and combining them into a single unit so that you could have Different output signals from your circuits. They're turning on whether the cell is producing nitrogenase or is secretion competent You can start to build up that that function So just to conclude I Start out by describing some of the genetic challenges that Confront you when you try to transfer a genetic system from one organism to the next and so we created this Process whereby we go in and are systematic in stripping out all of the native regulation and by doing that We create a system that's highly modular, but we lose the activity And so we have to go through this process of reoptimization or to recover the the wild type activity back Which then allows us to go in and be systematic about transferring it to a new host and trying out different Different combinations of parts in that host to reoptimize the activity So with that I'll conclude and take any questions Ocala My thinking is very similar to yours and perhaps because we both have background and chemical engineer And I think what presented was very clear and very nice Result, I would only say that it's important also to start with elements For instance, there are better promoters than probably used that are regulating from not Not existing in nature because I think the best way to regulate promoter is turn it back and forth when hurting I Yeah, and I didn't have a chance to talk about it, but actually our promoters as well as our our ribosome binding sites are Are completely synthetic and actually were designed themselves by computer algorithms, so We know the And because we're using phage polymerases. It's an ultra simplified very small promoter and If for us we need this like you described if you flip a piece of DNA So if you flip to promoter into a spot that would then be a very hard on and off in our case What we like to do is have a system that's completely off until we have the controller as a phage polymerase And so the phage promoters themselves both were engineered by computers and they themselves have really no activity Until you put in the controller that then has the phage polymerase is that then allow that system to be turned on and controlled and so the the point I'm making is that everything except the amino acid sequence itself of the final system We have was was completely by design So even a ribosome binding site not only do we know the strength But we know the strength because we have a biophysical model that calculates the free energy of ribosome binding to the RNA or RNA polymerase binding to the promoter and so on so we can really Decouple for that part exactly where it gets its function its contribution Yes The Yeah So one thing that's interesting is that for this and a lot of the projects in the lab We're actually driving more and more away from how the natural genetics are encoded And actually the next generation of refactor systems that we have Looked just nothing like biological organization So one thing is we've gotten rid of operons all together so that every gene is under monocystronic control its own promoter its own terminator Then on top of that we've we have insulators that are very good at transcriptionally isolating each one of those units and Separating the function of the promoter in the ribosome binding site so you can independently control them Whereas right now you can't And so you've got these highly highly modular Subunits going through the system that really just look absolutely nothing like the the native system Which what emerges from evolution is this kind of bubblegum and sticks just whatever worked next kind of putting it all Together and so you get these redundant functions and overlapping and so on and so forth in a big part of being able to engineer those systems This is strip all of that all of that away In terms of like the interface with how we think about it and directed evolution I actually came out of a directed evolution lab where you apply random mutagenesis to an enzyme and then Identify function or a metabolic path or whatever so we we definitely do that type of thing the difference here is that If you you can't do it to multi gene systems effectively So you could take a system and randomly mutagenize it and kind of select for improved activity But you couldn't take one of these clusters move it into a host and randomly mutagenize it and hope to just find an individual mutant That's gonna gonna work And so our the way our system works is that we put all of the information we have Into the to the program it creates the constructs Each one has the same amount of design information. It's a little bit But that doesn't mean each one's gonna have the same function So we can then screen and then based on that screening information Try to extract to new rules for how the system is composed and then put that into the next round of Design one thing I didn't show for example is that if you're systematic about breaking up the gene cluster We find that none of the genes have to be in an operon together except niffy and niff N And gee if you really break that pair up you run into trouble So the next round we build we say well, we'll make everything monocystronic because that's easy to control But gee we better keep these two genes together so that we were more careful about their regulation And so you learn systematically these types of rules that go back in and then help you with the next round of design Yep That Okay, yeah, so So there's there's two answers to that One that's highly sensitive that we don't know how to deal with yet and one that's less so so the the particular one showing this type of project where we have a large multi-gene system and really the parts that we're referring to our ribosome binding site strengths so if what matters is the ratio between proteins then you'd have to expect that those Those ribosome binding sites would go sort of out of rank order in the new Condition to the extent that it severely affected your your function and we're using phage bloom raises and so on so in that sense There's less of an issue. However the synthetic regulation, which is something I didn't even go into and it's not like synthetic sensors And circuitry saying when things should be turned on are highly sensitive to condition and so one example of that with the nitrogen a system is That it's very strongly ammonia repressed and when we eliminate all the data for regulation. It's no longer sensitive to ammonia But when we put it with our synthetic controller plus or minus ammonia it causes the Expression levels from the controller of the regulators to change which then changes the overall Activity of the of the pathway so you can go in and genetically fix that by tuning the expression level Higher of the polymerase than you had in the other condition, but really what you want is a system that maintains You know a set point control over the amount of polymerase independent whatever condition it has So what we find is the sensitivities that you're describing are mostly on the synthetic regulation side and less on the pathway architecture side So So that's actually a big current challenge. So One of the libraries I showed very quickly is sort of said something happened here And that's an example where you could go in by eye and we could see what happened and and rearticulate that as a rule Sometimes we're able to extract statistically from the data things like the NIFE and Combination that I was saying and then put that back in a rule the real challenge We've been having which we're not close to fixing is If you create a three or four megabase library where you diversify 16 genes in all different ways and very complex combinations By eye and by expertise and even statistical methods can't figure out What's going on? And so you get crazy rules from machine learning algorithms and so on and so where our focus has been is actually Not just creating the power the algorithms that allow you to go in and work out that data But actually design the constructs themselves in such a way that they allow you to extract the information when you're done with the experiment So and even old ideas like design of experiments applied to genetics and that type of thing actually become very very powerful So as another part of our next generation designs are Designing it to then allow us to test many questions in parallel Such that the algorithms are able to pull out certain classes of information from the data set Yeah Well any any allosteric issues would be exactly the same so there's no difference between the refactored and the and the Non-refactor system because it's the same proteins. It's the same everything's the same from the Right so in the native host we should be able to recover activity when you transfer it There's certainly going to be Situations where you wouldn't be able to do it for a whole number of reasons But there are a lot of examples of projects where just getting any activity then allows you to So you get a small amount of activity of nitrogen ace in certain target organisms That really gives you the foothold to then push that forward or if you're scanning for natural products like chemicals They're being produced by native organisms You only need a small amount for that initial transfer and then you can run the directed evolution or whatever it is That you're going to do to then optimize that system back up But so for us it's really about getting Increasing the probability that you get some function immediately after transfer. Yeah So what we found so we only build the genes once Because we found as long as you get like a reasonably expressing gene that it's you can control the expression levels Pretty much wherever you need with the synthetic parts. Well, I actually don't when we were taught I was by Bob Landick about this earlier that The cut on optimization as a means of controlling expression levels is really terrible So basically it's that there's something about the gene that's screwing you up if you have low expression levels And once you fix that then that becomes a the substrate for for being able to swing three different levels of activity and so So for us that's it once we have 16 genes that we know work Well, we can we don't have to go back and create new for new versions of that as part of the process Yeah Yeah It's enough where you can grow slowly on so you could do a selection experiment for example, in fact we have


The thing that really jumped out to me is the Yankees did everything right and the twins seem like they did everything wrong. This is a hundred and one-win team and I never thought they'd be swept as good as the Yankees are. I thought they'd show up a little bit more than this. Well, you would assume that, you know, as many home runs they hit throughout the year that one game they would bust out. But, you know, I agree with the guys in the studio that they just look like a better team. The Yankees look like a better team. They caught the ball. They made the plays. You don't know how deflating it is as a team to be down. And every time you have an opportunity to score runs, somebody makes a great play. And the Yankees, sometimes their defense was a little shaky throughout the year, but so far in the playoffs it's been great. Hey guys, let's talk about Aaron Boone and the job that he did with the bullpen this entire series. We know last year in the postseason maybe a little slow going to the bullpen. I thought he managed it brilliantly tonight. And you can see exactly what he's going to do the remainder of the way. And he kind of gave you a hint over the first two games. So if he gets four innings out of a starter, he's thrilled. He's not going that far without a Vino if he's not throwing strikes. If Kainley's not sharp, he's going to take him out. And as he did in game one, he was prepared to get nine outs, or six outs, not nine outs, between Britain and Chapman. That's exactly what he did today. The only thing that stopped it on the first game was they got more runs than he brought in Jay Hap. But that looks like it's going to be the formula. They want more than an inning out of the last two guys.  I mean, Mariano used to get six outs, five outs at times. So Tori would use him in big games. I mean, this is a game where you've got an opportunity to close out a team. You use your best guys. And right now you're waiting to hear what happened to Britain. But I don't think he had any intention of Britain finishing that eighth. He was going to get to Chapman anyway. Paul, I'm sure there were some seasons where you headed into the postseason and didn't love the way your swings felt at the end of the regular season. Didi Gregorius was in that situation this year. And now you look what he did in the division series. And I'm sure he feels like he is ready to go and play for the rest of October. What did you think of Gregorius' swings tonight? You know, a lot of people question Gregorius and Tori's coming in the season that they didn't have great September's. It means absolutely nothing. As soon as you get to the playoffs, it's different. And I'll tell you another thing. Now that this first series is over, look for Sanchez. Look for Stanton. Guys that seem to be getting closer, they could have big series next series. So as a player, you close the book on this, you enjoy it. But now you get an opportunity to do something big in the next series. And I will tell you, there's added pressure on Didi Gregorius because he's going to be a free agent at the end of this year. Now at the beginning of the year, even when he was coming back from Tommy John surgery, he just assumed that he'd be back. But Glabor Tours did so well filling in for him. And then he struggled since August. So this is kind of his, you know, plea. All right, I want to stay here. Watch what I do in the postseason. So this is doubly big for him. He wants the Yankees to win and he wants to put on a show for himself as well. Michael Pauley, tell me a little bit about Severino, especially that second inning bases loaded. Nobody out. What was your feeling at that time? Jeff, I thought that was the game right there. I mean, the twins had an opportunity to make a statement. They're in front of a loud, raucous crowd. Bases loaded, nobody out. Didn't seem like Severino had great command. His stuff was pretty good, but the command wasn't there. And when he got out of that jam, I thought to myself, game over. Oh, there was no doubt. I mean, that was their opportunity to get this crowd involved to, you know, to beat the home field thing. And I think it showed up the next inning. Cave strikes out and then he dies for a ball in left field. That's almost desperation that he had no chance. And that's just frustration of not coming through a big situation. Again, the Yankees in every aspect of this series outplayed the twins.


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This is Global Connections. And the question we are asking is, how concerned should we be about Hezbollah? How long will Hezbollah constrain its attacks? And what will it do? Remember that it is directed by Iran as all these other non-state actor, terrorist groups on all sides of Israel are doing. And for this discussion, we have Rupmati Kandakar, a geopolitical strategist to join us. Welcome to the show, Rupmati. Hello, Haji, and thank you for having me on it. Thank you very much. Well, let me say that the one thing I want to sort of lay down in this context is that you have to look at all the dots and connect them. And I don't think a lot of the media does that. And they accept press release disinformation from various sources. And you really have to connect the dots. You have to use critical thinking to figure out what's going on in the Middle East. So we have Hamas in Gaza. Hamas is a non-state actor, but it controls Gaza. It is the government of Gaza because Gaza is a failed state. And it in turn, that is Hamas, is in turn controlled by Iran. It is trained, directed, and it is armed by Iran and coordinated with other non-state actors. So one of those other non-state actors is Hezbollah, which is in Lebanon, which is another failed state controlled by non-state actor terrorists. Okay, and they are not particularly concerned about human life. And they use human shields pretty much the same way Hamas does. Okay, and then there's another one, it's Yemen. Yemen, you'll have to agree, is another failed state which is controlled by the Houthis who are non-state actor terrorists. And they are controlled, surprised by Iran and armed by Iran. And so what we have is now three of them there. And we have the same process going on in Syria where the US is fighting in Syria. So this is sort of spreading out some more and Israel has to cope with the non-state actor terrorist groups who are all sworn to do two things.  That's what it's about for Iran. And it seems to be getting worse. And Iran wants to keep the war going for as long as possible. So it is not particularly interested in a positive result from any ceasefire negotiations. And maybe that explains the positions that are taken by Hamas and the others. It wants the war of attrition. It wants to push the US out and isolate Israel. And it's doing that. And Israel's policy is to respond but the US policy is to perseverate. So the question is, the question I put to you, Muddy, is the US doing the right thing? Is Joe Biden doing the right thing in terms of dealing, in this case for this discussion, with Hezbollah in Southern Lebanon, in all of Lebanon? Jay, in our previous programs, we have discussed the role of Iran in this war. And now when you asked me this about Joe Biden, he's drifting from his policies, Jay. And when, like you said, the delay that is happening in this war is helping the other side rather than Israel. And Jay, in these three movements that you mentioned, all three are Pan-Islamist resistance movements. All three are militant outfits which have taken out a political role to keep their relevance. And all three have a single-soul mission to wipe Israel off the map that is reiterated by their master of finance, that is Iran. And these three don't have the military capability to deal with the state. So the deal in these poking tactics is Hezbollah's far more powerful than Hamas. And it was involved in rocket-firing against Israel from the October 7 terrorist attack on Israel, Jay. And they kept on doing this. And recently, like... I think you should emphasize the point that Hezbollah started lobbing rockets and drones into northern Israel immediately after the October 7 atrocities. They didn't wait right away. Yes. And Israel is not drawn when the defense minister says, we will be bombing into Lebanon because they know the threat from Lebanon Israel and southern Lebanon, Jay. Hezbollah has this strategy. The modest operanda is of firing rockets. And the recent one that happened was in Kyrgyzstan, Shimonah, Rivasat al-Alam, and Aviviam. These cities in, I don't know the part of my pronunciation, but these cities in Israel. So if a foreign terrorist outfit is bombing your city, don't you have the right to self-defense? These words like genocide, apartheid, all these words come into play. I think the word that should be emphasized is the right to self-defense by the only democracy in the Middle East against a terrorist attack which is funded by these militant outfits, Jay. Just taking on a political, minuscule political role doesn't make them a political entity, Jay. They do carry out targeted militant... They do hurt civilians. They have this... What is that? Their support for Hamas, Hezbollah, Hamas is not a secret, not a secret in the media, but it's not reported that way. Yeah, well, you know, so let me go back to Joe Biden. He doesn't seem to be taking strong steps to support Israel. He did it first, but then you saw this backlash in the US, this anti-Semitic backlash on many, many, many American campuses. You saw these protests and all that. Protests that favored Hamas, it was really extraordinary that all of a sudden, after atrocities, within days after atrocities, there were these protests involving many tens of thousands of people in the United States and for that matter in Britain and elsewhere in Europe, you have, on the one hand, you have the atrocities and on the other hand, you have this propaganda war that you mentioned. So Joe Biden is swinging from one side to the other. A couple of days ago, we had Kamala Harris get up and say, well, we have to have a peace settlement and all that. So my question to you, our group body is, does in your view, does Iran want to have peace here or isn't in Iran's interest and within its mission of not having peace? Jay, the Islamic movement in 1979, the revolution, Islamic revolution, and Hisbulla's Genesis was in 1980, so 82. So this is not a coincidence, Jay. Iran has funded and given birth to Hisbulla as a proxy for them. They fight the wars that Iran cannot come directly inside.  They want Hisbulla to do their work. And when you blame in international politics, they will say, we didn't do it, Hisbulla did it, so go attack Lebanon. This is the right thing that the trajectory that they follow and media does not report this, media has taken a narrative that Israel is the oppressor rather than the oppressed. These, if you count the number of attacks, if you count the number of states which are threatening Israel every day on a daily basis, it's phenomenal, Jay, how much they have to face. And Jay, Hisbulla will discuss the Shiasumi divide. It is a very interesting point in those factions that they keep aside all this in just in order to attack Israel. And Jay, what do you call a militant outfit which has the sole aim to wipe out Israel as their manifesto from the beginning?  And you never get to the result, you never get a peace. And if they really wanted to have a peace, wouldn't they release the hostages? Wouldn't that be instead they're killing the hostages and the hostages are dying over 150 odd days now. So I don't think anyone can treat them as sincere in any effort to have peace. So yeah, the other thing I wanted to mention is that week, and this is a part of the discussion with on YouTube with this fellow, Amin Dean, who is a former, he's a Saudi and he worked for Amin Laden years ago. He's very experienced in the whole Middle Eastern Islamic divide you were gonna talk about. And he explains what's going on here. And it has all the indications of a perpetual war, a war in which we have attrition. And after all, Israel is surrounded by all these people who want the river to the sea and destroy Israel. And that means, of course, Hamas, the Yemenis, the Islamic Jihad and Hamas in West Bank. The Russians now are in the golden heights. Who did I miss?  And they have invested 5% of their entire population, of their entire Israeli population is fighting. How long can they continue to do that? What kind of an economy can you have? And for reasons that are really not clear, well, that are clear, I guess, is that Congress isn't giving him any money and letting him wither on the vine. And so how long can Israel keep this up against a very well-funded, well-trained, well-armed group of terrorists that are in all its borders? So I feel that the war of attrition doesn't go well for Israel. And I don't know how long they can continue to do this. They haven't stiffed up a little.  This is all citizen soldiers and they're not participating in it. Your thoughts? You're so right about this, Jay. How long can they continue is the biggest question. Cease fires, all this is dealing tactics and they have replenishments in their forces. One on one comes, they're like a pack of hyenas who are hunting and they come one on one, one after the other, one after the other. And they are refreshed and they are reinforced and they keep on attacking Israel. And Israel has to deal not only with these follies, they have to deal with international media, international pressure.  When do you concentrate about the economy? When do you concentrate about being a state? How long can they give up? And that's what courage is all about, isn't it? It's just about surviving and somewhere you need divine intervention, I think, which they do draw their strength from. You know, I may sound, you know, it's out of my position to talk about this, but it's always divine help that Israel can stand up to these nations. I don't think any other country could have which took so much of resistance, Jay. And for so many reasons. Can you imagine what would happen in the US if somebody attacked us like this? Remember 9-11, you know? And we, the Israelis were attacked. We didn't start this. Anyway, you know, I'm concerned about the Lebanese border and Hezbollah in the sense that they do have more sophisticated weapons than Hamas. They have more soldiers than Hamas. They're closer, I suppose, to Iran. And they have been lobbing rockets and missiles into Northern Israel, increasing distances since October 7th. And so the Israelis are saying, well, you better not do that anymore. If you continue to do that, we're gonna have to counterattack you. In the meantime, Israel's taken 80,000 people of the cities along the border there. And evacuated them to other parts of Israel, whether they're living with family, friends, or hotels, what have you. It's not like there's a place waiting to take care of them. They have to be relocated in the evacuation. And so when Israel says you better not do that anymore, how serious can Israel be? Because if Israel actually goes after them, it's gonna have a big fight on its hands. And then it'll have two fights going on. Or more. Or maybe three fights. And can it afford to have a major fight on the border of Lebanon? So I wonder when they say, you better not do that anymore, whether they're really saying, we'd rather not have a major fight with you. What are your thoughts? Yeah, Jay. Trying to avoid this. Jay, one thing about them is that they are funding. And these rockets are so... I use the word again, targeted. They have, Israel has got this diverse aims and objectives to stop this border to clean Gaza to protected citizens. For them, it is just attack Israel, attack Israel. There is no other aim for them.  They're determined in depth and focused. Now, Hezbollah makes rockets in the name of Israel. I mean, they don't have stockpiling or they don't have any other mission. They don't have anything to protect their borders. It is just targeted towards hammering Israel. That's the only thing. Aim, sole aim for that. And Jay, a little bit of background on them that Hezbollah is a Shia militant organization. Hamas is a Sunni militant organization. And Shia-Sunni divide is huge in the Islamic world. We don't know about it as much as we just give a fleeting response to it. And Lebanon is 30% Christian, 30% Shia, and 30% Sunni. So the legitimacy and the authority that Hezbollah gets is by showing that they are fighting Israel and they try to garner support in the Shia-Sunni faction of Lebanese population. But they don't get that kind of support because the Shias, Sunnis don't support Hezbollah, but because Hezbollah is supporting Hamas, which is a Sunni organization, they get that support. So it is your enemy, my enemy, we are friends. Maybe I don't know that kind of tactics happen in these militant organizations. So they forget the Islamic divides and they go forth for militant aims about Israel. That's very, very dangerous. And Lebanon was a Christian country 30 years back, if you know. Yes, well, I mean, Israel's surrounded by failed countries. Think about it, every single country, I would accept Egypt from that, but Egypt doesn't want Hamas in its poorest because it treats Hamas as a terror group. So every one of these terror organizations is in a failed country, meaning that the failed country can't, doesn't have the resources, people, or political control to do anything.  And the common denominator is Iran, motivating them all to attack Israel. So I guess I'm worried about what position the United States should take because somehow, see if you agree with me, Lebanon is a key feature here because if the war on the Northern border, Sharia, Shimonah, if they are attacked and if people who have evacuated, 80,000 people have evacuated to the south, and they are attacked because these missiles and rockets can reach over the zone of evacuation and into central Israel, if this becomes a big war, what happens then? And what is the United States gonna do? And this is a surrey question, but what is the United Nations gonna do? How about nothing? But how does the calculus change if Hezbollah really does attack Northern Israel? Right now, they just have these sporadic attacks with missiles and rockets that they're not using the full measure of the armaments that they control. They just wanna keep Israel off balance. That's what they're doing and have this continuing threat. But if it got into a real shooting war there, how would that change the calculus? How would it change the need for American involvement? Jay, Israel's war tactics from the beginning of October 7th, terrorist attack on Israel, we saw that before anything being proved, before anything being, they continued their attack and resistance on the Hezbollah from the Lebanese front. They knew that this is the front which will keep on poking, so they knew they had to keep it on bay. And till now, they have kept them resisting them. So Israel is very clear about Lebanon to hold them back because they keep on poking. US as an ally, as we keep on talking about this, that it's the hegemon of the international system and to have a vantage point in the Middle East or to have the existence of Israel as the only democratic state in Middle East is of vital importance to the United States. And to keep it going to support it is not only a matter of military or civilian importance, it is also humanitarian because there are six countries who are trying to destroy a country out of the map. How long can you have that? And not only the country, that's an entire race which they want to make it vanish of the map of the human existence. And that cannot happen tomorrow. They will climb down on the next target. After Israel, it will be another target. And Jay, when you have this much of the Islamic resistance movements, if, and God forbid they never achieve the target of eliminating Israel, they will be a breeding ground for more terrorism. They will start planning more terrorist attacks in all the democratic states, the Sharia law, the Sunni law, this law, undermines democracy in every which state. So Israel is not only a state, it is also the beacon of democracy. And Jay, we lessen the value of democratic issues because if we don't keep Israel at the forefront because see functioning liberal state in the Middle East is a rarity, it's a single one. We don't have many Israel, we just have one Israel. So if you want to preserve this kind of outlook or thought, it has to be, the ally has to be in, the allies in fact have to be in full support and wavering support in the Congress, I think has to be overcome. It has to be a overt policy of supporting not a covert one because right now there is entire, there's an overt attack on Israel. So it needs overt responses in every which way. And a small cutting back Jay is very dangerous because Israel is being, Ukraine is just facing Russian aggression. Let me take it, if you want to compare it, just Russian aggression on one front. Israel faces these kind of attacks on six fronts, seven fronts, and with a gorilla warfare tactics which just overwhelm the traditional military apparatus. And Jay, in that, there is a growing anti-semitic sentiment all over the world where Jews all over the world are targeted. So this is the spilling over effect is more dangerous for Israel. So this is a very complicated situation Jay. And support from allies or non-allies is very, very important. But I like one thing that Israel is very determined in its task and it knows how to fight without support, with support, and just for, like you always say, this line is yours, existential crisis is gone. So it needs, it has to, it has no other choice. And any- Let's go ahead. Yeah Jay. Well, let's turn to the US for a moment. I mean, we are now, let's see, voting begins, absentee voting, you know, begins in September, some states for the November election. And we're, let's see, April, May, June, July, August, five months away from that. And maybe seven or eight months away from the election itself. And it seems that Trump has said that he would like to see Biden fail. He would like to see Biden fail in everything possible. Immigration, for example, wants to see him fail there. He wants to see him fail in Ukraine so he can blame the failure of Ukraine on him. He gonna fix Ukraine, Trump says on day one. He'll fix it by giving it to Putin. And of course, he would like, you know, this war to continue in the Middle East so that when he gets elected, he can make peace. So the likelihood, given the fact that the MAGA GOP is effectively controlling and blocking aid and a resolution of the immigration crisis on the Southern border. It's in Trump's interest to keep everything in suspense and not to allow Mike Johnson to solve any of these problems so that he, Trump can blame the Democrats and Joe Biden. So I suggest to you that we aren't gonna see an American initiative to help Israel, not in a major way anyway. Joe Biden may provide them some weapons, but nothing as we would have in the bill that he submitted or cause to be introduced. So if let's assume that nothing does happen where the United States gets his act together and supports Israel the way it was supposed to, what happens politically here in this country? I mean, A, it makes Joe Biden look weak, maybe in some ways he is. And B, it makes Trump look stronger. And that would affect the election. Would it not? Your search? Absolutely right, Jay. He plays to the drama, to the tea. And Jay, American foreign policy, irrespective of the precedence, like we say, it falls in line. And Israel has always been an ally, irrespective of Republican or Democrat. But Trump will want to take the upper hand or showcase that he is at the upper hand. And Biden's failures will be Trump's triumphs. That kind of mindset that he's got, I hope it does not move towards any dangerous zone for Israel. And he continues to keep the support for Israel going irrespective of natural domestic politics. Because this is something beyond our borders, but it will affect every Jew within American borders. And election promises, election promises on Israel, they are very muted about it, because we know that the general sentiment amongst the people is not that well educated. They do not have a very clear idea of what is the Israel-Palestine conflict. They see 200 protesters waving the Palestine flag. They think Israel is bad. Nobody goes into the nitty gritty of international politics or what is happening, really happening in Israel. They go with the mob, they go with the masses. So coming out with these kind of statements, pro or against Israel, they will always restrain themselves, these two presidential candidates. They will keep it muted, but underlying current will be towards being an ally for Israel. That's what is my thought about Israel. And at one point of time, Israel is still playing conventional military weapons. Israel is still valuing a civilian life. Israel is still targeting military targets in Lebanon, in Gaza, everything. At one point of time, they will force Israel to give a more sophisticated response to these mini rockets. I'm sure about that. I hope so because what we saw in the Hamas attack on October 7th is that if you throw a swarm of rockets and missiles and drones against Iron Dome, Iron Dome can't handle it. I don't know if Israel solved that problem. I don't know if the U.S. has provided technology to deal with the swarm problem. But we are all learning and they are learning and Iran is learning, Iran makes drones. They supply the world with weapons, including drones. So it leaves me with two questions I want to ask you.  Is that they're the sympathetic with Hamas? And just like the Houthis, they're going to continue to attack Israel in the case of the Houthis. They're going to continue to sink ships and they actually did sink the Ruby Mar just the other day. They sank it in and cut the cable and it did terrible environmental damages. They are going to, they will stop attacking Israel if Israel agrees to a ceasefire. And of course, Israel is not going to agree to a ceasefire unless there is an agreement for a ceasefire. And of course, Iran really does control whether there is ever going to be an agreement for a ceasefire. But let's assume there is a ceasefire. Do you think that Hezbollah will stop attacking Israel? That will no longer be a threat. Israel, Hezbollah's manifesto is to eliminate Israel. How will they stop? It's the truth that Israel has accepted. And just see it instead of just looking towards only Gaza and from where the attack was coming, Israel knew that the attack is also from, the threat is real from Lebanon. And so they did not leave that front. And this kind of alertness about Hezbollah is always, always, always very necessary because there is somewhere there is always in Islamophobia that loyalty is very rare. They do stab you in the back when you're not looking. There's always a rule that you always hit from the front. An enemy, you should deal with the front. But Islam, in Islam, they do stab you in the back. That same came from there. The interesting question is, we always, we look forward and we wanna examine whether there are variables that could come in from stage right to stage left that we haven't anticipated and that would change things. And as I was asking you about the political scene, the ever-changing political scene in the US, whether that would change things. And I suppose what happens in Ukraine could also change the, what do you wanna call it? The global calculus, the global environment around these wars. But one thing I don't understand and I wanna ask you your thoughts about it, and that is why is Russia building a military force in the Golden Heights? What is Putin doing? Doesn't he have enough going on in Ukraine? Yeah, Putin has never been away from Middle East politics. We have seen his presence in Syria all throughout. He has supported, he has... There was one article I don't remember. It was a gift to Putin on his birthday, something like that and I don't, I'll send you that link if I find it. And Jay, Middle East politics is a big, big distraction from Putin's aggression every and each time. When there is aggression over here, you have the planes falling and assassinations happening. You have something spectacular happening in the Middle East. You have some other underhand dealings happening in Russia. So he uses it as his playground. And Iran has been an ally of Russia, so always. So this kind of, and Iran's outlet in the Middle East, no, in the global politics are these militant outfits. Not only just, they don't have an international standing if they don't support these small, small militant outfits. So Russia sees this, they, how do you tell it? They heard these outfits because nothing concrete comes for Russia, nor they have to indulge in tremendous amount of spending. But having these groups to their advantages is always beneficial for Putin because Putin's foreign policy regarding Middle East has always been to have a hold. That has been his, they don't want, Israel is a stronghold ally of the US, undoubtedly. So how does Putin come in? He can't, he can't come in directly. So he supports these militant outfits. It's like a schoolyard, a schoolyard fight. You know, in one corner of the schoolyard, you see all the bullies gang up on someone. You got to get over there and join the bullies. That's what he's doing. That's my opinion. And one thing that's related to that is that you have the EU and you have NATO. Now, Israel is not a member of NATO, we should work. But they are focused on Ukraine. I'm not saying they're helping Ukraine. Maybe they should help Ukraine a lot more. Ukraine is running out of soldiers and it's running out of ammunition. It's running out of morale. And that attacked the other day on whether it was intentional or by mistake by Russia against the prime minister of Greece, Anzolensky and Odessa. That was pretty threatening. So we have Ukraine is in a bad place for the lack of support by the US. My question to you is, how much can Israel count on support from the EU, countries of the EU and for that matter, the countries of NATO? Have they been helpful here? Would they be more helpful? You think if there was a fighting war, I mean a really hot war on the border with Lebanon, with the Israelis, the IDF and Hezbollah? Jay, at one point of time in international politics, I always thank it that they are not that effective organizations towards their opinion or to implement their decisions. They have decisions which are just binding, the guidelines, not binding. And they don't have military prowess to enforce it because the kind of discussions that are going on in the EU and are not worthy of the Israeli defense state. And if you see that they have a lot of pressure from the internal domestic politics, the right wing, left wing is going on over there. So they have those kinds of issues that come in the EU decisions that come in. And NATO is a good force, but we have the hegemon supporting Israel. So that is a balancing tactic in that. But as international organizations, the EU is a false short jay. And Netanyahu had told a long time back that this war from Israel will move to Europe. Now, when he said that, he had a very clear-cut idea that when these immigrants go to Europe, there are already existing terror cells in Europe which watch the politics in Middle East very closely. And at will they can have these hotspots in Europe which are vulnerable to attack. They're not going to stop. And they keep on getting stronger day by day. Tell me a European street which was the same 10 years back. We used to go on holiday. We used to love it. We used to love the European culture. We used to love the history behind it. And now it's defaced. There is a total cultural invasion. I don't mind immigrants. I don't mind people coming in and staying in for survival.  For me, truly, personally, European-ness is destroyed at a very fast pace. 10 years from now... It also has an effect on the politics. You know, this anti-Israel politics from migrants, from Islamic migrants in Europe. It certainly has an effect on elected leaders in democracies there, don't you think? Right wing, left wing is so acute. You know, Netherlands, Denmark, these places are coming out with covert expressions of hatred. And Jane, right now, these terror cells regroup, come back, and they will be reinforced with these migrants who will move from the Middle East towards Europe. And when they have European environments, European facilities to... To honor support or, you know, to establish their own identity over there, you think they will leave? Lebanon was a Christian country 30 years back. We can't let go of that fact that it doesn't take long for them to completely take over. And immigration, migration for survival was a different cause 10, 20 years back. Today, it is just for regrouping, reinforcing, and oppressing and taking over. This taking over from migrants is going to be a big problem in 10 years from now. Writers about migration cannot write about anything else rather than what will be. Netanyahu was perfectly right when he said that the problem moves from here to Europe. He gave the destination, he gave the location. That way. Well, you have to be concerned, even if they want to be isolationists in Congress. The fact is that we could have terrorism here. You suggested that possibility before. I want to throw one last possibility at you, Umadi. That is this. We've looked at all this coordination, direction weaponizing from Iran. It's not going to stop. It's going to get worse because, you know, what did that fellow, I'm in Dean say, he said, Leading with weakness will get you war. Quote in that video on YouTube. Leading with weakness will get you war. And, you know, I think this will continue and Hamas will get stronger and more bold. And so will Iran. And so all of these terrorist organizations, they are pretty bold right now. But if you say that Iran is controlling Hezbollah, and if you see that Hezbollah is very weaponized and has sophisticated missiles and drones that has not used yet, have not deployed all of its weapons, it seems to me there's a possibility that Iran is holding them in reserve as a strategical matter.  So my question is, do you feel that, you know, from the four corners of this conversation, I feel that Iran may be holding Hezbollah in reserve like as a military battlefield strategy to use at the right moment. Do you feel that too? Do you think so? Absolutely, absolutely right Jay. In our previous programs, we do have discussed is that the October 7th terrorist attack, they knew that it's not going to destroy Israel in one shot. It was targeted towards provocation, expecting a retaliation and then a long drawn war strategy. It was just a provocative attack on Israel. They knew that they will have a retaliation and this kind of, they are thinking beyond us, Jay. They're thinking 10 steps further. And Iran, like the point that you said, Iran is holding Hezbollah in reserve is such a valid point because right now they have not shown anything. A few rockets is not what they have. The Shaheen drones that is Iran supplies to the world, they can put the entire thing on Israel. So these strategies are still waiting to be unraveled and Israel is at a greater risk now than it was before because they are just regrouping and reinforcing their ammunition. And the target is getting stronger. The target is determined, but their enforcements are getting stronger and stronger by the day. Now, when you talk about Russian presence also, it's Afghanistan was just Russia and US in Afghanistan and the local this, but this is a far more complicated situation in Israel. Israel is alone, fighting and grappling for US support. Ally, when you have totally, totally antagonistic atmosphere surrounding it, it is encircled by this and Jake constant consistency in the attacks which are against Israel is a very hard hitting feature of this struggle. They don't stop. They don't stop in the night. They don't stop in the day. They don't stop any time. So 24 hours alertness is difficult to maintain. A country can maintain one line of a border or one front, but constantly having to defend your country 24-7 is a daunting task. And that kind of, it's very daunting.  There has to be action and there has to be decisions right now, results right now. Yeah. And finally, when I get out of this, I think we're out of time, but let me offer this thought to you is that support by the U.S. and soon and right now becomes increasingly more important to Israel, to the survival of Israel with all of this encirclement you talk about. Well, thank you, Rootmati. Rootmati Kandekar, global geopolitical strategist, really appreciate this discussion. We'll follow up because this is a moving target. I hate to use that word. And there's more to come. Thank you so much, Rootmati. Now I like it. Thank you so much. Aloha.