Day2-PRECISION FERMENTATION FOR SUSTAINABLE MANUFACTURING OF BIO-ACTIVES AND INDUSTRIAL BIOCHEMICALS

Day 2 | GIAN Course on “PRECISION FERMENTATION FOR SUSTAINABLE MANUFACTURING OF BIO-ACTIVES AND …

good morning everyone sorry for the delay so yesterday we finished our conversation on cellular Agriculture and precision fermentation right when we were discussing the topic of cellular agriculture we talked about the a cellular Productions or product Productions we also talked about the cell biomass being itself a product when we were discussing about the a cellular production I talked about transforming the host or providing the capability to the host that it can produce the product of Interest right now how do we do that how do you transform your host or provide it with the capabilities to produce the product of your interest you have to make some modifications right it’s like asking if you want to change someone’s personality you first have to understand the person itself right so keeping that in mind so this part what we just said like to change the personality of the person you need to know the person itself that’s what sets the basis of all our conversation that we are going to have today it would be more about understanding what happens or what changes we can make to the bacterias or to the host organisms specifically like yeast bacteria would be the eoli and how what are the tools that are available with us all of this that we are going to discuss fall under three different topics that what we have the synthetic biology the systems biology and then you have the enzymes and the engineering enzymes so we’ll start with synthetic biology now in my own personal Viewpoint synthetic biology actually encompasses all the concepts that we should be discussing today if you’re talking about system biology it has uh the parts of system biology in it uh if you’re talking about enzymes it is going to have the parts of enzymes in it so you might be getting a bit more of that feeling that you already are able to understand enzyme engineering or engineered enzymes in here do what the system biology exist now what you see here is the definition for uh the synthetic biology so synthetic biology is defined as the design and construction of new biological entities such as enzymes genetic circuits and cells or redesign of existing biological systems so as we started our conversation what want the equal ey to be able to produce a specific product if I know how the eoli functions what is the metabolic pathways that are followed within the eoli system I should be able to tweak the eoli or The Tweak the metabolic pathways in such a way that I can get the product of my interest do you agree to that how do we tweak it fals within synthetic biology what are the tools what are the engineering tools that actually we use will fall under this particular topic now it is different from uh the tradition of molecular and cellular biology as the focus is on the design and constructions of core components because we are only focusing on the design and construction of the core components it differs from the molecular and cellular biology so we will looking at metabolic pathways part of the enzymes we’ll also be looking at genetic circuit so whenever keep whenever I keep saying genetic circuit it’s like you have the genome you know what the genetic uh what the Gen code is and what each and every Gene is going to represent like what it will translate into so if if you know that there is a gene sequence let’s say and every section of the gene is going to translate into one specif number three or should be producing number three more than rest of the other proteins like one and two so if I can repress these sections of the gene and only allow this section to translate I would be only getting my product of Interest so there are ways to do it right how do we do it those are the tools that we going to discuss on the left hand side you’ll see the basic uh the road map that is basically used in the synthetic biology the DBL or dbtl is your design what is a b build test and learn in as an engineer when I’m talking about as an engineer engineering involves designing things right so you always design first we build it a prototype we test the Prototype we learn from the Prototype and only then we’ll go into the bigger format of it right so that’s what exactly used at a mole ular level so we have taken a macro level idea towards a molecular level idea now so the goal is to engineer cells to produce something useful and I will be showing you some of the examples we’ll be discussing a bit more in detail about those examples so for example if we have to look at the bacterial production of insulin and if we are using host you remember when we were discussing the host selection is a very important parameter when we are looking at uh production of a specific product of Interest now what was the criteria for the host selection can I repeat some of those criterias non-pathogenic should give more more quantity so you should be able to produce more of your produ H and and ease of Downstream processing or ease of extraction or ease of recovery what else understanding of different metabolism Pathways so if I had to say it in a Layman way it would be that you should be aware of the host capabilities in such a way that you can make the modifications that are required and cost effective cost Effectiveness will come into picture only then right if you’re are not aware what the host can do it it will be a very costly affair right good so I’m happy that you listen to the things that we were discussing yesterday so same thing now the same concept now let’s say if you have to produce insulin and you’re using eoli as your host what would be the very first thing that you’re going to do you’re going to design a genetic Circuit of the eoli or of the insulin production so what you have is let’s say so this is please excuse my drawing if this is your eoli this is your genetic material your DNA of the ecoli okay now there are two ways of uh producing your product of Interest now if the insulin now where do you get your insulin gene or the gene that is going to translate into insulin where will you get that insulin uh Gene of insulin from what would be the source who requires insulin humans right if somebody has diabetes insulin is basically produced to break down the sugar right so you need the insulin so you will get the genetic information from the human genome that has already been looked at right so you can take out the genetic sequence from there now you can take that genetic sequence and you can pass it on into the equoli using a a vehicle now that vehicle is called a plasmid so this is your plasmid now what is the other unique property of the plasmid so plasmid contains the sequence of Interest so that it can generate more of it right as it will replicate it will keep on generating more of that particular sequence and then eventually that sequence will translate into the protein if that protein is my uh protein of interest when the equalize growing right now what other properties now do you think every equoli that I have my plasmid into will be able to run the plasmid or the plasmid will work in that equalized system I might not be able to frame my question properly what I’m trying to say is let’s say if you have 10 equalize so you have provided the plasmas into all right now will all of them be producing insulin at the same efficiency or will they have different efficiencies like some of them might have lower efficiency some of the strings might have a higher efficiency some would be like mediocre which one will you choose and how will you choose it depends on the plasmid in what way like what is the structure of a plaset the circular I was not asking that like what is what would be the component of the plaset so you have your Jone of origin and then you have your antibiotic resistance why do you have this screening of what yes obviously so if we have antibiotic resistance then we can grow it on and we will like get the bacterias in which we have our insulin and in those which we don’t have our insulin so can plasmid also be used for selection purposes so is that a tool to add selection properties so can you repeat your question so if you have the not all bacterias would be doing that but that one would be so if I am only interested in the one which is producing my insulin to the maximum can I use this as a selection criteria for like for maximum we can’t use it like like everybody will be producing some or the other amount of the insulin for maximum property we can’t use that we cannot use that but what if like one which is not producing one which is producing we can do that we can do that right very good so for this type of selection it cannot be used but when you’re uh selecting it from the microbes that don’t have the plasmid or they’re not producing your insulin you would be able to use the plasmid as a selection criteria that that antibiotic resistance property that is provided by it now so you have your host so either you are going to add the plasmid to provide that insulin in there or you can make a cut within the genome of the bacteria or the eoli and add your Gene of interest to produce your insulin which one do you think is much more effective and easier so either I make the changes to the Genome of the equal itself or if I’m adding that external DNA strand using a plasmid in the equoli which process would be much more effective and which process would be much more easier to do using a plasma would be easy why to some extent we are sure that it will be producing the insulin now you have answered it but you have answered it in a very different way I like the answer actually it’s correct so the other thing is like when you are incorporating the specific genetic material in the eoli DNA itself there is no guarantee that it might get transcribed too it might be in a sequence that that material or the Gen but having it in the the DNA sequence itself doesn’t guarantee that so the efficiency or Effectiveness might be less but there is no way until unless you do experimentation to actually State whether it is better or not okay now so there are two ways of doing it either you add the plaset or you can add it into the DNA sequence itself so now you have the you have to build the genetic circuit and put it into the bacteria so I have built the genetic circuit like the your plasmant which is your genetic circuit and I put it inside the bacteria and then you test how much insulin is made or how much insulin is being produced and once I have optimized the one the strain that is actually able to produce most of the insulin I can take that strain and start growing them into the in the fermentor now that is exactly what this particular uh flowchart or the set of diagrams are representing so you have your human insulin Gene DNA you have your plasmid Loop of the bacterial DNA which is there in the eoli now plasmids were actually found in eoli they are separate to the the DNA of the eoli and these plasmids were the ones which were providing some unique properties to the eoli and that’s where the interest for us as scientist came into for the plasma that hey we can use these as materials to transport it can be an amazing Vector so now you have inserted your uh Gene of interest for the insulin into the plasmic Loop you pass it onto the bacteria you grow it you select it you put it into the fermentor and you start producing the insulin now that once insulin is produced or enough insulin is produced you can Harvest it and then purify and then you get your medicine your insulin okay now this is called your recombinant DNA technology technically this is your recomended DNA technology and also an important part of precision fermentation now synthetic biology actually encompasses the tools that we used to make that change of how to produce that insulin what were the tools that were used to move the or to cut the genetic sequence for the insulin from the human genome put it into the plasmid put it back into the equalized cells Let It Grow the growing part would come into the fermentation aspect but the rest of the things before we get into that is your synthetic biology but if you don’t understand the metabolic pathway or if you don’t understand the microb itself it will be very difficult to make the changes that we wanted now that part understanding the microb as a whole what happens within the microb would come under the systems biology and if instead of insulin I was let’s say developing or producing a new enzyme or I was making modification in the DNA sequence of the host to produce an enzyme of my interest with my capabilities that I like that enzyme to have that would be your enzyme engineering all three technically can be considered as one topic but there are three different tools that are used now so the approach that we use with respect to the design build and test is the designing which is your biological circuit or a system for a specific function using computational tools the build uh consists of a system by assembling editing and installing your genetic material so that’s where the plasmine comes into picture then you have your test the system functionality that once you have added whe whether you are able to produce the insulin or not that would be your test and once you have done that you put it into your firment system you can learn how the system is is performing what sort of modifications you need or what optimizations you need to produce more and more of it now this is your road map for your uh synthetic B ology now let’s look into the goal how the same thing that we had here the design the build the test and learn how we implemented for our insulin so the very first part where we are designing the insulin precursors so designed by uh designing sorry designed by finding the gene of the human insulin precursors and optimize model for the equal expression and then you move it on to build the gene sequence including the insulin precursor genes through synthesis and assembly and then you test your circuit in cells by measuring the amount of insulin that is being produced and then learn how the system works and what optimization is necessary for growing the insulin in the fermentation process your dbtl can Al is part of your Precision fermentation but before that so where we were looking at starting the culture so preparing the culture the host and then moving it on into the lab scale five lit 10 lit so you’ll have the Shaker flasks so all of these experimentations are done first in the Shaker flask to make sure that they are working then you do it into a small scale that will be 2 lit or 5 lit bioreactor and then you will do it in a pilot scale and then you’ll move it on to your commercial scale the one of the best examples that I can actually give with respect to application synthetic biology and an amazing application of synthetic biology would be the antimalarial drug artemisin now artemisin is produced in a plant atmia anua that particular species which is throughout the throughout Asia and Africa as and it is used as a tea and it press juice is used to treat malarian related symptoms like and chills it’s active ingredient is your timis which has been developed as an antimicrobial drug or sorry antimalarial drug and is used worldwide if I have to get this artimis to make my drug from the plant itself what would be the process that you will follow the process is there on the slide so you can just explain me that but what would be this the process that you’ll follow you’re going to grow the plant and extract extract it from then you have to come up with the process or extraction process Chop Chop protocol the chop it pre-re pre-at and what else so pre-treatment okay so grow Harvest post Harvest I’m not going to do Chop Chop oh okay chop chop and and then clean cleaner something uh Chop Chop can include the cleaning part also okay uh let’s say pre-treatment uh pre-treat much more important one and then you are going to do the extraction yeah it a powder form or something and once you okay why Chlor form no Powder powder form oh the powder form okay okay f extraction and then you’re going to put that into a powder form so for that you have to dry it right right and are you using a conventional oven or you’re using a freeze dryer based on the budget based on the budget okay I will Define the budget here because we are going to spend some time on this particular slide so okay so you have your budget you have your drying you have your powder form and then you do what you give the powder to the people to eat you can give for the next with the microbial we can use as the feed feed for you’re using this powder for what uh so for the next stage for the for oh that the the the thing that you’re seeing about no Tim missing comes out after extraction so this particular powder can be used as is like so that’s you have so many examples coming out from patanjali actually I gave it with respect to my work what I am doing so that’s why I give the okay now I I I realized that but I’m just so you have the chop chop you have the free that’s a very mechanical way of saying it I really like it now somebody can explain me in the bological OR biotechnological perspective what the chop chop will mean [Music] I will I’m I’m from food engineering background okay and I will try my best okay so uh for example if whatever the antimicrobial drugs uh therei sorry H antimalarial drugs there is a lethal dose to uh like a specific dosage is required to uh destroy the okay uh like get the effect so uh first of all for whatever uh drug which is available which is in the encapsulated form or compressed form that is available in the market so accordingly uh we can extract it and we can encapsulate it and the controlled delivery can be done and uh it should be stable in the pH in our good and uh it should be uh like wherever it is required so I thought like definitely it should enter into the bloodstream so definitely it should be stable until large intestine okay so it should be stable like we have to encapsulate it in that form to deliver into our drug so most of the anti microbial forms I think it’s in encapsulated form it’s available first of all pelletization after that we can encapsulate it yeah so you’re talking about Downstream once you have the powder I was asking if you there’s a way that you can explain what Chop Chop was so you had the the grow you have harvested it you cut down the tree mated it got a powder out of it of the tree itself uh and then you’re doing pre-treatment pre-treatment means reducing the moisture content okay right yeah yeah or by uh making a fine powder so that you can increase the surface area because to improve your solvent extraction efficiency you need to have a very high surface area right so you’ll do that and then you’ll do the extraction process now what would be the solvents that you use for extraction it depends upon uh even different solvent having even different polarity so this particular if it is a polar compound you’re going to use a polar solvent if it is a nonpar nonpolar you’re going to use nonpolar solvent right let’s say if it is a non-polar compound uh we will be using nonpolar like what would be the non-polar solvent that you’ll use for extraction purpose go ahead you can answer it’s okay there’s no wrong answer in this particular session so please go ahead chloroform acetone hexane okay dither or what else so almost all of these are your non-polar solvents are they good for you so they’re always going to leave a residue right when you’re doing an extraction with chloroform and acetone and xn they’re going to leave a residue on your powder after the drying Pro but when you’re looking it from the practical perspective you might not be able to go into microwave assisted extraction or ultrasonication I’m not going there that’s not the topic of our conversation I can give you a 4 hour or 10 hour lecture on that but my question here was that once you get the powder you’re basically looking at a highest Purity because you’re going to use it as a drug right so you want to get rid of any traces of any solvent that was basically used now this whole process is a very long process and the solvents that are you are going to use are also not very good for your health and you will need a very costly Downstream processing methodology that will get rid of the traces of these organic solvents and you’ll get your product of best quality or the highest quality do you agree with that right so practically it’s possible but I will not like it or I will not like to use it for uh let’s say mass production whatever like to have in that case is to identify which part of the plant xenome is responsible to produce this arine and then I can use that put it into a genetically modified eoli or a yeast or a fungi grow it produce it on a larger scale right that would be the interest and that is exactly what people did because malaria is a major major issue in Africa it’s also a major issue in India and at one point of time there were very limited availability of drugs and there were the drugs that were available were very costly so synthetic biology actually provided the possibility to develop Tim at a very cost effective way or in a very cost effective way and it made its availability across the globe and now that is also used as a very first uh first line of defense so we are using this as the first treatment that you give to make sure that uh the person is cured of malaria so one of that example now would be that the production of amorph hadine which is your precursor and its conversion to di Hydro artisic acid which is again the precursor for arine so emorine is being produced in the yeast now yeast as a source because before this they did in eoli when they did that in the eoli the overall yield was less they made changes they looked into other host they found yeast they modification to the yeast they got much better yield so that’s how the host selection will also change because you sometimes it’s not that you can decide on the day one itself that you know what this is the best host that I can use for my genetic material to grow you might have to do some trials onto it so you’ll do with the Eola you’ll see whether you able to improve the overall functionality and product uh production of your product of interest if it doesn’t work then you try if you have understood the metabolic pathways in the East and if you can make modification into the yeast you will start growing into that because the fermentation process will change what sort of fermentation system do you think you will need for the yeast because in the later courses or the later lectures that we have tomorrow and day after we are going to talk about batch reactors we going to talk about the FED batch reactors aren’t we so we’re going to talk about these type of reactors and how to design these type of reactors I’m not saying physically but mathematically so what sort of bi reactor you’re going to use for which type of host matters the most so when we are going to use yeast and for that you only need the input so that you can produce your product I can use a Fed batch where I can only give the input there is no output in a bat reactor there’s no input there’s no output once you put everything in you close and that’s it it’s like uh when you cook rice in a pressure cooker you put the rice you put the water close it on the stove done and then you wait it to whistle that’s your batch reactor good example now same rice you added less water initially but it’s in a it’s an open pan you’ll add more water as the as it is boiling right so technically that becomes a Fed batch so you’ll add the nutrients for the cells to grow so for the yeast we generally use fed batch so this one was done on a Fed batch reactor and what you will see here this particular diagram here and I really in if you can read this particular paper it’s really interesting paper uh they did a genetic modification into the into the genome of the yeast and they compared to their previous uh cultures or which were used earlier the amount of emine that was being produced was pretty high the amorin and also you can look the arisc acid is pretty high so your y151 is your Generation 1 this is your generation 2 so there were two different generations of the the microb and the plasmids that were being used or the processes that were being used within those micros to grow and to produce these precursors so in the generation two you get a much better yield so that’s how we keep on improving our productivity we have to continue so we can start with the eoli we might get to a certain level we’ll then change it we’ll try to another host we might be able to get something else as part of of your uh Gan series you’re going to do two case studies one is based on a plant protein we are going to have some form of discussion today in groups what source will you choose like what host should be there and what will the process and where will you get your sequence from how will you get that sequence from all of that will be discussed in this class today so in the synthetic biology uh the systems biology and the enzyme engineering so we I will give you the background for it to build on your case study so well it’s a good example right so like the producing the malarial parasite sorry not malal parasite malarial drug please excuse me on that you’re not producing the malal parasite but do you know how the malaria parasite actually works you have heard people get malaria but do you know why do get why do they get malaria or how the how does that plasmodium falum which is the parasite impacts so when it comes inside what does it do where does it grow I’m not able to hear you so I’m so sorry RVC in your red blood cells have you heard uh sickle cell anemia what is Cle cell anemia your your red blood cells are in a CLE cell shap right and that is caused because of a genetic mutation people having CLE cell anemia cannot contract malaria so genetic modification can be good for you it’ll kill you anyway but not with malaria at least right but the point is these genetic modifications are very very important they’re very natural so microbes go through genetic modification in a very natural way so let’s say if you want to produce a specific genetic product or like like a protein but you want to make sure that it is is done in a very evolutionary way so you’ll make a small modification to the DNA sequence you will let that mutate from one generation to the second generation to the third generation and you’ll keep on checking if your protein of interest is being produced and in what quantity and the moment you reach the strain that is actually producing the protein of interest in the highest quantity possible you choose that but that has been a very evolutionary way of making that modification it’s a very tiring process it’s a long process but it’s a good process okay which I’m going to Showcase right now so like this do you have this any anyone has that or have you ever seen any marking at the bottom you’ll always see a marking like this on a plastic bottle right now now talic acid is the precursor which is basically used to make p so talic acid is a precursor which is basically used to make your p and this p is mostly used for your making the bottles and the Plastics right now if you’re looking at the conventional process you’re going to see that it is using Cobalt it is using manganese those are heavy metals and uh you would have heard about heavy metal pollution right in the soil in the water so if you’re looking at a process chemical process that are using these Metals you don’t want to use them because you you would like you prefer to change it it’s not environmentally sustainable process so we have to come up with something else but if there is a way for us to produce this PTA within a microbe I would love to do that because I can produce as much as I want or even more but what you have like in a conventional process in the normal regular Pro processing you’re producing in bulk and that’s the reason why it is only costing you very less it’s only a dollar a kg so the cost of that is pretty less because you’re producing in bulk using chemicals now the same thing was done using microbes and the efficiency that they got was approximately 6 G per liter which was equivalent to be honest it’s very equivalent or similar to what you would have got in a conventional process but when you see the diagram on the left it looks very very complex but if you have taken a course in Biochemistry you would be able to identify some of the components within that right can you identify those for me you have it is actually saying you have your glycolysis cycle you have your TCA cycle that’s your biochemistry 101 that’s the very first thing that you study in Biochemistry right now why do we need to have a gly colis cycle in the TC cycle who has this cycle here in this particular situation who has this cycle where is this cycle present right now within the within the micro within the host and the and the host is is there in the title right so your eoli is basically the host and the glycosis cycle the TCS Cycles are present within that now there would be a specific Gene sequence in the eolive that would lead to the glycolysis cycle that will lead to the TCA cycle that will lead to the other biochemical Pathways right once you have identified what each each and every genetic material each and every genetic sequence in a in a in a genome does so you can map the whole body but they all are interacting with each other it might so happen that once the gene 3 is activated that’s when only Gene six will get activated because it develops a precursor to uh to activate the Cycles or metabolic pathway for the gene six or Gene 7 right so those type of understanding understanding the relationship between the genetic um tools where you’re adding these plasmids these are the two plasmids here at the bottom so you have the origin and then you’re producing your product you’re initiating the production of your product so this is carrying your Gene of interest and then it is going to produce your product and that particular product the TPA is going to get released extracellularly and then you can remove or recover it so now we have moved from a chemical synthesis process into a biological synthesis process now Effectiveness might be comparable Effectiveness might also be less sometimes but even if it is less you keep on doing the permutation combination you keep on running it you can keep on changing the host you keep on playing with the feed stocks you would be able to improve the overall production efficiency of your product at the very end and this is where I actually I wanted to discuss what the plasmid looks like but we all were able to answer the questions beforehand so I’m not going to discuss what the plasmid is but it also utilizes the same design build test and learn process okay now what are the tools that on the right side you will see in the green so you have your engineering DNA the mo biomolecular engineering the host engineering and your data Sciences these are the tools that we basically use as part of the synthetic biology now for the engineering DNA you are looking at the synthesis now whatever we have discussed till now even since yesterday we have been always discussing about making modification to the host DNA correct cor now what if if I tell you let’s make our own DNA can we do that what is the DNA made of nucleic acids nitrogen is bases phosphate group and what would be those nucleic acids nucleic acids will be that um just give me the alphabet cyto 80 0gc so DNA is made of a t n g and C right so it’s just a sequencing right of a t g c t g a c and you can keep on changing it you keep on getting your double standard DNA or single standard DNA now that’s the idea so can I make it if I know if I have let’s say all my precursors as a building block right if I have my a if I have my T if I have my G if I have my C I can make my own DNA sequence so rather than making waiting for the host DNA sequence I can actually make my own DNA sequences so that synthesis will require some engineering tools that can be used to do that other would be the genome that is present in a host or in the micro or in the e if I have to sequence it to actually understand what is their atgc sequences if I can identify the tools that can be used to identify that sorry if I can develop the tools that can be used to identify that would come under the sequencing you have heard about the Human Genome Project right that’s where we were uh sequencing the whole Human Genome and in all mystery movies detective series specifically I still remember the one which I used to watch here see ID I hope you are aware of that one I love that one okay da that one yes okay so in that if you had to do the DNA sequence they we do talk about that later in their uh some of the serieses right so they’re doing the um they get the hair sample or the blood samples and they’re matching the DNA right how know how to sequence it you print and then you are able to match it at one point of time in history that was completely unimaginable you couldn’t even imagine that this thing is possible now it is possible earlier the same finger the DNA fingerprinting used to take weeks or months nowadays it takes hours because of the engineering advances that have taken place and those advances that I’m talking about are part of your synthetic biological tools that have been developed over the years the computational tools the mathematical tools the engineering tools all of this combined have given us the efficiency that we can actually sequence the DNS sorry sequence The genome and get and study the metabolic pathways understand how each and every uh genetic uh code is related to each other and what inter relation do actually they have with their phenotype typic pathway that understanding that relationship understanding is very very important when you’re working with synthetic biology then comes the standardization the assembly we can assemble all of these and even if you want to do the editing like the way we did in the plasmid so all of that comes under the tools of engineering DNA then some of the examples would be for the synthesis and sequencing uh at one point of time the cost that was involved in terms terms of looking at in late 1990s if you were doing the DNA sequences per bases the price per base what would be the base how many bases are there like how many okay what is the base is a a base what is a base pair a a a is a base pair and GC is a base pair so is a a base yes so identification of just a just T just G just C so the cost per base not base pair per base that is what I was talking about at one point of time it’s like $100 now is in like in sense we can do that so it’s pretty low right with the Advent of engineering with more and more advancement that we have had this whole thing has reduced now we are capable of doing DNA sequencing and the synthesis uh at a very low cost now you have the the single standard DNA if you want to synthesize those uh you use a solid phase chemistry uh which actually includes uh control po glass beads and then you are able to put that and then you can get your sequences done people like me uh when we do research and if we do need to have a sequencing done or let’s say synthesis done for a DNA uh I’ll say the one we used to have you heard about this term apar have you heard this ter what exactly is it single A single standard standard of DNA which might be specific to something specific right like we used to develop this after for microt toxins if I have to identify a microt toxin if I want to develop uh a sensing a biosensing uh technique I would be using this aper because they would be very very effective and my sensing capabilities would be the best in that aspect so to develop these short standand dnas rather than meet you use a process called a selects okay now that was completely unimaginable at one point of time so now it is very very easy I used to pay around $40 to get one aper done so the only thing that I had to tell them is that this is the base sequence that I need or this is the sequence that I need of my atgcs can you give me and I need a thle group in the front I need this in the back and they will make it the company will make it and then send it to uh they will ship it to us and then we will use it it’s as simple as that now companies like twist agilant they use uh silicon chip Technologies to make this single standard DNS so there are different techniques different different processes that are available for you to develop these sequences now other would be uh production of the double double standard DNS uh where you use the polymer chain assembly for shorter synthetic uh fragments and for the double standard genes are produced from stitching together those short single standard oligo using DNA polymer and PCR so PCR everybody’s aware of it you should have had demonstration I still remember when I was watching C at one point of time um they were talking about a PCR machine and that they showed the PCR machine but it was a microwave and he said like no this is a PCR it does this and I was so excited that this is how the PCR should look when I went for my undergrad it was a very different machine but it it’s fun like you get to know a lot of these things from your day-to-day life right so PCR can be used to make your double standard DNS and it’s a very common technique once it came into the market it has made life much more easier for uh molecular biologists then comes the genetic circuits now genetic circuits is an interesting part I’m not sure but have you seen some things like these when you’re reading some of these molecular biologies or uh genetic papers these diagrams I’m not I’m not saying anything else but just these diagrams now these diagrams are nothing this is on the right hand side you have your code or the language it’s like your a b c d e f so those are your alphabets and when you arrange your alphabets you get your words right similarly these symbols are used to design your circuit the idea was that it will resemble an electrical circuit so when you have venic acid and the symbol that is being used is to repress so the valonic acid is present this genetic sequence is repressed okay so if valentic acid is present only RFP is expressed the rest is repressed so the promoter is the start right so then the promoter this whole sequence will get expressed so only RFP will get expressed if the valenc acid is present so because it is repressing the rest of the process the vanr will not get expressed because you have the promoter here too but just because of the presence of the vanic acid it won’t work in this particular case in the second example you can see where you have the light source if the blue light is present what will happen look at the code on the right hand side look at the diagram on the on the left hand side your circuit look at the code and see what is happening if the blue light is present which one will get inhibited you have the promoter at the very beginning on the left hand side what is the first thing yf1 right followed by fixj fxj I’m so sorry if it is not clear is it not clear enough no the diagram is it not clear like are you are those alphabets legible no right I’m so sorry so the very first one is yf1 on the left hand side followed by fixed J then I think it is cdrb CDR a and CF okay so if the blue light is on there is repression repressor sign which one will get repressed yf1 and so your yf1 will get repressed even the fixed CH will get repressed with the innovation of the yf1 the fixed J will get activated okay Express the CD CDR a yes and if the fixed J is active you express the CL at the Varan that’s not CF that’s a very CL and if no CL Express CD R A so these are the that’s the language that you’re using so you’re using the symbols on the right hand side to write your electrical circuit so this is the very uh core of synthetic biology or when you’re doing computational synthetic biology they actually show you these things and this is something that they will Design the genetic circuitry and you would see a very the similar example here on the plasms you see the promote on the left hand side of the very bottom you should see there so you have the promoter and that promoter is going to use uh and switch on the X yl MSC and that will lead to the TSA mbcd and it will keep on going right now another aspect of synthetic biology is B biomolecular biomolecular Engineering in the biomolecular engineering what we are doing is uh the natural Macro Molecule such as the proteins they can be engineered to have new functionalities similarly where we were talking about the engineering enzymes so enzymes are proteins they are basically used for catalysis correct now if you want your enzymes to be capable of of for directed Evolution so you make one mutation you let it continue from one generation to the second generation to the third generation and then you are able to identify which generation actually has it now mutations question for you uh let’s say if somebody has CLE cell anemia one of the parents have CLE cell anemia will the progenies will also get the CLE cell anemia so if a parent has that will the kids also have it no every progeny will it get transferred every generation it might not get transferred every generation but mutation which is intent will get transferred every generation so if you want to kill the mosquitoes make a mutation the progenies will not and that is actually a research going on where they’re actually working on Chang the DNA sequence in such a way they have put up a mutation and that mutation once it get transferred it will be transferred from one generation or the second generation to the third generation but I don’t want that mutation I want it to evolve I want it to mutate by itself right so that would be that would be the directed Evolution I’m directing it so indirect evolution is like you just did and wait something might happen somewhere a direct Evolution where you’re directing the evolution to go in a specific order got the idea this directed Evolution please remember this term directed Evolution you might want to use it when we go into the case study too just a hint now directed mutation is basically used to make modifications to enzymes specifically so if you’re making changes to the proteins to the enzymes or you’re trying to develop new types of proteins new types of enzymes with different functionalities you generally utilize the directed Evolution process so directed evolution is used through rounds of sequence diversification selections for functions and amplifications now entirely new protein sequences not seen in future can be generated uh by through the denovo protein design so I can actually make my own protein even that is a possibility these days so that would be your denovo protein design so something that is not natural I can make it any questions tell now another aspect of biomolecular engineering would come with respect to novel metabolic pathways that can be constructed using retrosynthetic retro sorry retro retrosynthesis approach sorry for the slip of the tongue now retrosynthesis approaches anyone is aware of what exactly it is it’s a very interesting thing but uh it’s widely used in chemistry I might be totally wrong I see I’m I’m not I will never consider myself to an expert in system biology I’m an engineer but this is a topic that you need to have some background in so that you can understand the rest of the things that we are going to discuss in this particular course that’s the reason why I’m giving you this information but retrosynthesis approaches in my ideas let’s say if I ask you to make a specific food product you will ask me what would be the raw material let’s say right so if you have to bake a cookie you’ll take the flour you’ll take the chocolate chip you’ll take the egg and you take all the utensils that are required and then you can make or bake a cookie what if you follow the you go backward now you you know a product so let’s say I want to have a product a I know that a can be made from B and C C is commercially available but B is not commercially available so that means you can buy c as a raw material but B is not commercially available but you know that b can be uh produced from D but D is also not commercially available but D can be produced from e and f f is commercially available e is not okay but you know that e can be produced from G which is your commercially available now what are your raw materials to make a g g f and c rest all are not so we don’t show them as chemical reactions arrows we always show them as double arrows because that might be the the will give me D now this is proper chemical reaction now this D with some chemical transformation will give me B with a addition of C will give me a which is my final product now this is called retrosynthesis approach use that now with your metabolic pathways so you know let’s say that atile Co a have you heard about this term atile Co a you would have seen it in one of the now atile Co a is also a precursor for many many other uh products right so you know that Estel Co a is one of the process which is like one of the uh precursors for my particular product but that’s the final product but you need the cycle for the before you reach that IAL COA so you will go backward you start developing that cycle and you will somehow put it with your metabolic pathway your glycosis cycle that all this atile COA will start getting into that pathway the one of your interest or the one which you have engineered so that would be the biomolecular engineering aspect of it I am not asking you to be a champion in this I’m just trying to give you a basic idea that this is one way that we can do things is it clear to you so if you want to introduce a new metabolic pathways you need to know what like at least one of the components of your metabolic pathway should be present within the one of the metabolic pathways within the cell and then you can use that as a precursor to develop your own and then you can use repressors to suppress the rest of the cycle and all the atile COA that is being produced will get moved towards your Pathways and your product will only will be produced that is also possible that is your engineering here okay I’ll find few examples and bring it tomorrow on this now host engineering uh we have been discussing this as part of our Precision formation also so what are the different types of host that that there are and what changes do we do so either we can use a cell-free systems a single cell or multicellular organisms or even the biomass or the consor so the changes that we can make so what are the different types of host that we can use so what are the different systems so one would be the cell-free system where you are actually doing it in the lab in the in the chemicals itself so it can be used as a quick test for gene expression you grow your compounds to taxing for the cells and it needs to be extracted and purified for bacteria that’s where we get into the the fermentation side so you can grow well in bulk model for many many diseases we have used bacterias to look out how specific diseases are uh how specific diseases can be fixed that we can identify the proteins that are uh eliciting a specific response by your immune system by modif the host uh which is specifically the bacteria here it’s a procaryotic can’t fold many human proteins so if you’re trying to work with your human proteins in a bacterial cell it’s not possible because it is not capable of folding them uh human proteins are pretty huge too that is also one of the reasoning yeast on the other hand is pretty good in that so if you’re going for the proteins and enzymes yeast is a very good source or sorry very good host so it grows well in but milk can produce variety of proteins and often used for food and bread so in the yeast the best best example for the yeast would be the cchomes the other host that you can choose are your mammals uh so you have your human protein folding capabilities that are present because it’s us it’s our cells that we are using you can you use that for to test the drug therapies let me ask you a question uh the cell lines that we use the cancer cell lines what type of host are they will they fall within the mammal the cancer cell Lin depends helles and the Chell lines they are the Maman cell lines actually yes so I’m just asking so cancer cell lines will that fall with the Maman cell lines yes sir so cancer cell lines mostly when we are looking at for disease uh identification of drugs that might act against a specific disease within the humans we basically use the human cell lines right so those mamalian cell lines are basically used you will appreciate this fact the majority of the drugs that are developed are proteins and when when you’re working with proteins you always want to see how they will interact meleon cells are best to work with that because those proteins are mostly human proteins so testing uh drug therapies and they they very difficult to grow in bulk uh I have my own personal experience during my masters where when I was working on the cancer cell lines they will die multiple times I have to repeat that same experimentation like six or seven times to be able to do it a small modification like even like like if I’m moving it from one location to another location I basically just added a bit more of force I’m done restart the whole thing it used to be fun but not interesting that’s why I moved back into engineering but anyways uh plant cell lines uh they’re good for the photosynthetic Pathways and if you’re looking at varieties of compounds if you’re looking at generation of phyto compounds then plant cell line or sorry plant cells are very good hosts uh very few tools are available to work with them the way we have it for the mammals for the equaly and the yeast there are so many genetic engineering tools that we can use for the plants we don’t have that many it’s not a very well studied area uh it’s also very difficult because of the presence of the cell wall it creates another hindrance uh whole organisms that’s when we go for the mouse model and I’m going to now question your ethical aspect here how many of you have actually worked with mouse models if I may ask have you ever seen a mouse model like white mouses the ones which are used you know how we have to extract our things out of them right what do we do sir dissecting like extracted the pbcs peripheral blood cells out of it nonhuman way I remember when I was doing my masters at Mill uh one faculty in molecular biology and microbiology he taught me how to do it uh I was a TA for a course in microbiology and we had to demonstrate to the undergraduate students how to do that and oh my God like students will cry when we are doing that even we used to cry so that’s one for me personally it’s an amazing thing to work with them but that’s ethical aspect comes into picture so that’s why you will see majority of researchers are trying to identify host or identify processes in which they don’t have to go into this but sometimes it is necessary because when you want to see the phenotypic differences right that’s the only way you can see it you cannot see the phenotypic differences in the micro but you can only observe that in a living organism and that would be your mouth and it’s most difficult to engineer at the cell level now comes the end of your synthetic biology part now we have been discussing about the the cells we have talked about the DNA sequences the genetics you can the proteins uh directed evolution of the proteins now the question for you would be what sort of information do I get from synthetic biology like what are the techniques that we are using everything that we talked about they can be termed under uh an umbrella of something called as omix right proteomics genomics metabolomics transcriptomics and please go ahead majority are these so they will fall under the omix that develops the data that you get now from the proteomics you can get the information about the protein so if let’s say if I ask you to give me the structure of the protein I give you the protein name if you have to identify the structure of the protein where do you go pdb the protein database bank right so that would be the very first place I will look for a protein sequence right if I want the sequence and then the 3D structure or the 2D structure I will be or the primary structure of the protein if it has been crystallized and uploaded not all proteins we have not been successful in uh crystallizing all proteins yet but that’s an ongoing process as people are doing that they keep on uploading on the pdb so you’ll be that will be your very first source of information what if you want to study how the evolutionary trend of genetic sequence or the mutation took place what will you use or what do you want yes all the bioinformatics tools that you can use right application of that also falls under synthetic biology and you have to excuse me on that because I am not an expert in bioinformatics so I cannot teach you blast faster I would have done that as part of my undergrad but not when I’m doing my research I’m not working on these areas but these are the tools that are available now these will provide you the data that you can basically analyze later on to understand the metabolic pathways if you get all the omic data you can understand the metabolic pathway you can study each and every sequence of the genome and understand in uh the relationship the interaction between the different sequences one metabolic pathways and other metabolic pathways because sometimes the product of one metabolic pathway is the precursor for the other metabolic pathway so understanding that relationship having that as a map will help understanding and developing new strains developing new methodologies to develop or to produce your product of Interest using Precision ferment welcome back do you have any questions with respect to the previous part of the session that we had on synthetic biology if not we will get into the enzymes and and the engineering enzymes and we’ll also discuss about systems biology the thing would be that the the some of the concepts we have already discussed while we were having the conversation about synthetic biology so it might seem like a repetition and we are also going to uh go through some of the details about enzymes that you might be aware of if in case there is something that you would like to add please let me know we can add to to our conversation and if there is something that I am able to add to you that would be perfect okay to start with enzymes now what are enzymes and what is a catalyst Catalyst if I remember correctly from my chemistry is anything that basically moves the chemical reaction forward in a specific Direction so if you’re moving from A to B you use a catalyst it will make it faster but Catalyst are always materials that will provide their surface for the reaction is this statement true majority of times Catalyst are the ones that are providing the surface for the reaction to take place they’re also ones which impact the the uh the let’s say the activation energy that is involved in the reaction process process now proteins sorry all enzymes are proteins but not all proteins are enzymes that’s if I remember correctly that was there in the biochemistry textbook now proteins that speed up reactions in living cells to perform various functions like digestions muscles building and many more are one of the few of the functions of the enzymes that we are going to work with the one which you see on the right hand side is a lysozyme uh you you should have heard about egg white lysozyme protein so the egg white albumine or the egg white of the uh yeah sorry the egg white contains lysozyme as one of the proteins where else can you find lysozyme yes in your saliva right so lysozyme is a very important enzyme and it’s a very common enzyme now most of them are threedimensional globular uh proteins that means the tertiary and quary so when we were discussing about the the structure of the DNA we talked about the atgc but we we didn’t talk about the structure of the protein right now proteins are made up of amino acids so if you have amino acid a let’s say I’m just going to use a bcde for that so you have amino acid 1 2 3 4 5 6 7 8 uh so 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 now 16 Amino aets if they are arranged in a sequence this becomes your primary structure right once they start folding they will give you the the secondary structures what are the different secondary structures that you’ll have Alpha heles Beta sheets parallel and anti-parallel you will also have coils turns what else are there these are the major secondary structures that you will have right so all of these secondary structures so what defines a specific secondary structure that this particular protein sequence is going to give you uh secondary structure let’s say alpha helicis or a beta sheet what defines that what defines a specific protein would be produced in a DNA the sequence of the DNA right the sequence of your amino acid is what defines what sort of secondary structure you will have in a protein now when you take all of these secondary structures and then these secondary structures fold further they give you tertiary structure and when multiple tertiary structures combine together that gives you a quary structure right now all these structures of the proteins when you look at from uh from the very first slide if you say if you look at from the surface uh Dimensions so if you add surface to that protein you’ll see that there are groups these groups are mostly the locations where they are able to go for the lock and key mechanism so these are called your active sites might that might not be the active site in this particular isoy protein it might be here but I’m just saying the structure of the protein will Define where exactly your active site will will lie and what the structure of your AC side will look like now most of sorry can be obtained from Plants enzymes can be obtained from plants from animals and microorganisms through relatively simpler extraction processes it has been used for centuries in the food production process for cheese making beer making or sorry beer brewing and do leavening uh what other applications of enzymes do you see in the food sector clarification [Music] okay bitterness reduction so you’re talking about the sensory yeah it’s just adding the sensory value to it right can protein add texture meat tenderization what else is there the gluten protein that you find in the bread what does it provide blood uh like cross linking softening of the blood it provides that Clos linking and the network in which your starch is available right so that also adds to the texture of the bread so proteins are capable of providing you the textures they’re also not only capable of uh catalyzing experimentation sorry uh reactions but they are also capable of adding texture the sensory values to the food products and that’s why we are using them like a lot in the food industry from the pharmaceutical aspect enzymes are capable of working as a drug there is a medicine which you should be aware of unigy if you have an upset stomach that’s when they are given that unigy so it it helps you to make your digestion go well right so there are different types of applications of the enzymes because enzymes are the ones which are going to cleave the proteins into smaller bits and so that your body can absorb the amino acids any idea why we get allergies to protein then if we already have the enzymes and if they’re able to go and cut the specific protein into smaller bits why do we get allergies use the mic to answer the question autoimmunity like so you’re saying that when we D when we ingest the protein our autoimmune or our immunity sorry our immunological response against it leads to Allergy that that I’m aware of but yes sir like even certain protein even our body is consider as anti body so sorry antigen so for example egg allergy certain people egg allergies or for example in case of uh gluten intolerance like disease are there so it’s like immun like uh that that may create certain problem to those people like swelling swelling I know so that would be eological response to the presence of the protein my question I might not be very clear in that so my question is what part of the protein initiates that immune response is it a specific amino acid that initiat immune response receptors like proteins have receptors the body has receptors but my question is what part of the protein Elites an immune response you’re the biotechnology you should have a course in Immunology right I had that was a long time back try try see there is no right answer for most of the things just try your best and let’s say okay so PE you are aware of nut allergy many people do have nut allergies in one of the research that my graduate student did we figured out that I I sorry not my Gad actually a colleague of mine did we figured out that if you boil Peanut the allergenicity of that protein is less so the immune response that might happen is less as compared to if you are roasting that peanut so roasted peanuts will elicit a heavier immune response as compared to the boiled peanuts why proteins do get denatured at High temp roasting is at a way higher temperature because oil is at a very high temperature as compared to water CH there is a change in the structure right so similarly you can actually engineer your enzymes by because all proteins are not enzymes but all enzymes are proteins so because they are proteins I can change the structure of my enzymes by modifying the structure of my enzymes I can change their capabilities I can change the way they will behave in a specific chemical reaction okay that is what I wanted to bring our discussion back to but it is important for you to understand the structure of the protein to understand how the functionality of the protein is dependent on its structure if you change the structure let’s say if you change the sequence of the amino acid you will change its functionality but let’s say if you’re not able to change the sequence even if you modify the secondary structures you will modify the functionality of the protein okay so if I take a protein and let’s say I put it inside a microwave and I switch on the microwave for a minute because of the electric my Electro oxala microwave right oxala electric field my protein will go through push and pull because your Alpha helic structures are like a dipole so they have a positive and a negative so they will start rotating in the direction of the electric field and that leads to the turns and the twist turns and the twist so if you have like it’s like a wire so you keep on turning twisting it turning twisting you’ll break it if you use enough power you might be able to denature the whole protein so that what happens when you are working with the proteins with the temperature perspective so we do want sometimes for our enzymes to have the capability that they are not impacted by the temperature they should not be impacted by the pH because all these environmental factors if they are impacting our enzymes functionality we would like them to be safer we would like to make sure that we engineer them in such a way that they’re not impacted by these environmental factors at all that comes under the engineering enzymes now the structure of the enzymes is a global structure with a pocket which is called as an active site we already discussed that now active site is where The Binding of actives so with the residue takes place and which holds which help to hold to the substrate now acis site is generally or is generally less than 5% of total surface area so the design or the structure of the Lan that we saw even just one single group might be the active side rest all is not involved it’s only one section of the protein which is just maybe the 5 to 6% of it of the surface area which should be involved in the activ actively involved in the reaction process so change in the shape of the protein affects the shape of the active site and the enzyme function so if you take your enzyme put it inside a micro take your enzyme put it in the boiling water take your enzyme put it in an oven the moment you will add heat the protein structure will change the active side structure will change if you change the active side structure it might not be able to bind so those are the things that happen if you if there’s a mutation that takes place and your enzymes are not capable of binding to a specific substrate anymore then that particular mutation is working against you if you come up with a mutation that is going to give you an enzyme with an active site structure that will actually work in your favor is where I’m I’m going with the directed Evolution so if you want to change we would like to have those type of changes to to change the structure of the enzyme itself and you can only change the structure of the enzyme by changing the sequence or subjecting it to external stress like heat pressure or even Shear mechanical Shear that might help change the structure but those things are outside the scope of our conversation we would only like to work towards the changing the primary sequence that primary sequence change will give us a different change in the structure of the enzyme now these are some of the classes of enzymes you have oxidoreductase transferases hydrolysis liases isomerases Li gazes so they all have different uh functionality so oxid will go for the Redux reactions transfer ises for transer exchange of certain chemical groups lies would be on non-hydrolytic Bond cleavage isomerism would be conversion of isomers from Cy to trans or trans to Cy if I remember my chemistry correctly uh liases or synthesis would be synthesis of two molecular substrates into one molecular compound using ATP hydrolysis some of the properties of enzymes which are of importance for us would be their absolute specificity some enzymes if they have a specificity to specific spefic substrate we would like to maintain that or we would like to develop that type of specificity in our engineered enzyme for that specific subset so that we can get the product that we want at the very end the group specificity would be if there are structurally related groups it can act on both the structures uh Optical specificity would be to work on the optical isomers and the geometrical specific specificity would be uh specificity towards CIS or trans forms like fases or interconversion of fumic and malic acids the other properties of the enzymes would be the collidal nature of the enzymes the catalytic nature we have already discussed that it can be affected by the temperature and pH now these are the most important parameters that we are going to work with now when we were discussing about Precision fermentation when we were discussing About Cellular agriculture or whenever we had the conversation about the fermentation as part of cellular agriculture or Precision fermentation we didn’t talk in detail about the process optimization we did discuss that the process optimization is required but what are the process parameters in the fermentation that are involved so you have a fermentor let’s say we are just going to draw a single uh a simpler fermentation system so you have your impellers you have your Sparger so which is your O2 what are the process parameters here the speed and rotation of the impeller would be there one process parameter O2 concentration let’s say would be another one the temperature can be another one I have a temperature Pro I have a pH probe right so I can change the pH I can change the temperature now will the enzymes get impacted by the impeller speed just guess will enzymes to get impacted by impeller speed maybe but it’s highly unlikely but maybe because impellers are not moving like regular fans the ones which are running around here but they are just pretty slow it’s mostly to maintain the homogeneous mixture of the medium right or to make sure that oxygen is diffusing throughout the medium so when we come into the design of the reactor you will see when we talking about a mass transfer how we want the oxygen to be diffused throughout the reactor and that’s where the impeller speed the design of the impeller comes into picture the other aspect the ones which we can control and one which we which changes as the product is formed or the substrate is consumed is your temperature and pH so the temperature and pH can impact whether your reactions are going forward or it is stopping completely or it’s going backwards right so enzymes being impacted by temperature and pH is not something new for the fermentation process if the enzymes are involved there so it is a very important process parameter so these two are very important process parameters and we always like to see if the enzymes or the proteins that are involved in the fermentation process are actually capable of running at the process parameters that we have chosen so if I choose let’s say a temperature range or of 50 to 80° C my enzymes and the proteins that I’m working with or even my product should be stable at this particular temperature my draw metal should be stable my product should be stable and the whole process the chemical reaction that is taking place should be stable at this temperature range pH mostly would be in a fermentation process what is the general pH is acidic alkaline or neutral acidic so you’re going less than seven how much less than seven do you think you go like 3 4 it’s go 6.5 6.6 that’s still neutral so you maintain somewhere near the neutrality so you want to maintain that so some of the products that are formed will add like if ethanol is a product that is formed it is going to increase your acidity of pH or decrease it it will increase the acidity right so that means it will lower the ph and when it lowers the pH what happens to the microbes they die right so that’s why your substrate now had is acting as a inhibitor see you all are aware of the design of reactors I don’t need to teach that now so that is exactly what happens when you’re looking at the substrate being a product being produced and sometimes the product will act as an inhibitor for the growth of the organism now that’s where the enzymes also come into picture if I’m able to maintain the pH by producing other products to balance counterbalance it so there are things that can be done now it also speeds up the reaction by loading the activation energy and it should not impact your end product the one which you’re working with now the synthesis of enzymes as we were discussing in synthetic biology you have your DNA so DNA gives you RNA or mRNA and this mRNA gives you proteins right I if I remember correctly it used to be like one sequence is red then another there’s a gap and then another sequence that is red and then that gives you the MRNA am I correct it has been a long time that I have refreshed my molecular biology but anyway so you have your DNA sequence you get your mRNA from the MRN you are getting your proteins right now this is where your ribosomes come into picture right now the information is carried by the DNA which is clear and the amino acid forms to make the specific enzyme so the sequence of the amino acids that we are going to work with Will Define the type of enzyme that we are going to have now there are intracellular enzymes and extracellular enzymes so intracellular enzymes are synthesize and retain within the cell the example of those would be the reduction reaction in mitochondria so some of the dedu that are generated there extracellular enzymes are synthesized in the cell but secreted to work outside now when they are working outside outside the cell or outside in the cytoplasm where so when we say extracellular is it outside the cell always intracellular can be in the cytoplasm but it’s still within the cell okay so examples would be the digestive enzy enzymes by pancreas to work in dudum now many of these extracellular enzymes are also used to digest uh some of the food molecules that can be used by the cells for the growing for the growth for their food and uh you would be amazed to know that these the capability of cells to produce enzymes extracellular enzymes is what uh make them very very interesting like specifically microbial uh some of some of the microbes they can grow on plastic they can actually eat plastic there are fungi that can eat plastic how are they able to digest plastic it’s not like they’re eating like the way we eat what are they doing so they are producing enzymes that are basically breaking down the plastic into smaller units that can be absorbed and used as a source for the growth of the fungi or the bacteria so that’s why enzymes play a very important role if you’re looking at from the environmental application also not just from the food application but from the environmental application these enzymes play a very vital role so any like currently there is a huge amount of research that is going on in this direction where they’re looking at identifying enzymes that are produced by different novel bacterias that can actually eat plastic in my own research group we are working on microplastics so we are trying to identify microbes or we are trying to identify processes in which we can actually remove these microplastics from the environment or from the acous environment so enzyme understanding how enzymes can work or how we can engineer these enzymes might help us in future in developing products let’s say we would be able to develop a product through fermentation just like a like a dried enzyme powder and you see that there is a water body you know that is contaminated with uh microplastics you just spread that powder on that water body and you that particular microplastic would be degraded over a period of time uh you should um do you have pets at home if I may ask so people do have pets like specifically I have two cats at home and pets sometimes due to their behavioral issue they will pee outside on your rug or on the floor and that smell which is there to get rid of that smell there are products that have been developed n Naturally by using enzymes that will break down that urine and the odor completely so that’s a biological way of getting rid of the odor that’s the biological way of uh developing a cleaning agent you would have heard about these uh enzyme based detergents oxie which is used aial it has so they also have these enzymes that actually work with the fiber to get rid of the Dust where are those enzymes coming from where are those enzymes produced in bulk you can actually produce them through fermentation process or through chemically but that’s where one of the application the common application that you can see so the mechanism of enzymes that is basically your official lock and key model which is the most common that we are aware of so it was proposed in 1898 by EML Fisher so you have the active side which is rigid and there’s no change in the active side so the actic side if it is an l shaped it will remain L-shaped you cannot change it so only subset which are L shaped which can fit into that active site will be able to come and bind to the enzymes so substrate is a key that fits the lock of the enzyme and that’s when the chemical reaction will move forward similarly there is another one which is costance induced fit model now this is where you’re basically forcing the active site to take the shape of your substate Atri side might be of different shape and size but you’ll take the shape of the substrate so it’s flexible so which one would be better this one or the rigid one for larger application you want something which is much more flexible for specific application you want something which is very very specific right I’ll just give you that as a hint this will come in your case study think about it later on now enzyme engineering why do we need that why do I need it to modify enzymes we need to modify enzymes for the industrial and other applications so designing enzymes by changing the amino acid sequences through recombinant DNA technology that’s where your directed Evolutions comes into picture is one way of uh modifying it now enzymes exist in nature are taken and modified with random mutagenesis sorry this is direct mutagen sorry Evolution the other one is not my mistake please and you start with a protein from the public database which is your pdb you focus on its active SES and you place the reagent or the substrate in a way to catalyze the reaction now you would have heard in the bioinformatics uh about drug delivery or no drug Discovery and this drugs Discovery is through drug design in biof formatics they do that a lot what do they do it what do they do there sorry so are they designing drugs that will fit the enzyme or they are designing enzyme that will fit the drug think think once you’re ready for your answer you can raise your hand we’ll give you the mic and then you can answer that so are you designing your enzymes to fit the drug or you’re designing the drug to fit the enzyme you would have heard this term right sir yeah sir actually we are designing the enzyme because uh there is two kinds of docking when you do it like rigid docking and flexible docking yes so when it comes to flexible docking we particularly select a particular uh like of the pting structure we select a particular reason where it’s binding so when you take rid doing is take take a like a huge section of the enzyme but in uh this one flexible docking we take a specific section me we decrease the area which uh to where that phys chemical compound which will later on serve as a drug is binding to the enzyme so your enzyme structure Remains the Same or you’re you’re changing the enzyme structure and if you are changing the enzyme structure in your docking process how are you doing that uh are you using different types of enzymes or you’re randomly deciding you know what it should it will look really good if it looks like an s or it looks like a or b how do you do that so basically uh during the first uh we they go for blind doing Blind docking it like we see which particular section or which chain is the part chemical compound is binding yes then we select that particular section we decrease the area and to increase the efficiency where it’s actually binding so uh I believe it’s uh uh it changing the structure of the enzyme so mean to increase we you’re trying to increase the specificity specificity yeah to in before that you have your enzyme you have your drug multiple drugs yes you do a blind yeah docking right whichever fits yes sir whichever fits and then you start looking studying the active s yeah that is part of engineering enzymes yes sir so your bioinformatics that you are studying right now is also synthetic biology it’s also part of your engineering enzymes I just wanted to break that so because you under students of biotechnology some of the graduate students here so you should be if you are able to go back to that particular course refresh some of those parts you will get a much better idea okay so why should I modify enzymes we would need to modif enzymes to produce enzymes for industrial other application that was already there and the other part is to improve the stability and the activity of the enzymes as he was discussing about uh the drug Discovery aspect if you are changing the structure of the enzymes you might be able to uh bind it to more drug there are some specific drugs that will work if if you have multiple components that can work against your or sorry multiple drugs that can work against a specific disease if you can make the modification in such a way that all those drugs can work then you the cost is less it’s not just one drug is available in the market you can have multiple options right so increase operating time of enzymes and the duration to minimize the cost the other aspect of Eng generable enzyme would be the properties what are the things that we are basically modifying or what are the things that we are trying to modify so you have your uh the PH range under which they will act the thermal stability of it the solvent uh tolerance these are all the operational parameters and the substate product tolerance now these things will get used when we are working with the fermentation process and we are using these enzymes in the fermentation process and if you’re looking at from the the reaction perspective itself you’re looking at the different substrate ranges that it can work with the specificity of the enzyme to a specific substrate the novel reactivities like the reactions that it can generate the co-actor use and the kinetic properties how quickly it can run that specific reaction or how quickly quickly it can catalyze that specific reaction the other would be the immobilization and collocation now this is basically based on where exactly you’re locating it I will not go into that that is mostly on the structural basis how easy is the access to the active sites so if you want to make your enzymes much more accessible you can engineer them in such a way that active sites are outward most of the time so that you can bind to the substrate much more effectively and much more specifically so that will improve the overall reaction process okay what would be the easiest way to do that if I give you an enzyme just for our conversation so let’s say if I give you an enzyme which and those enzymes will have the polar surface area non-polar surface areas right and the active SES will have the polar sides and the non-polar sides because not all molecules that are on the surface of the protein are polar in nature some of them would be non-polar in nature so when you look at the very first diagram that we had uh this one so let’s say all the whites are the polar all the blues are the non-polar so you have sides different sides it might be the other way around but you have sides so my question there is sorry coming back here I got lost so you have your enzymes with the polar and the non-polar part how do I change it like what what what will I do to its environment that it might change its surface structure itself what can I do to the enzyme what is the inherent property of a biological system a biological system always wants to be in the most stable State possible right structurally so if there is a balance between the polar and the non-polar sides for us if I change the pH of the surrounding the protein will try to rearrange a structure in such a way that it is reaching that balance again right if I change the temperature and the pH I’m forcing it to change faster maybe but changing the environment the pH specifically because isoelectric points are involved there right if I change that it might change the structure of the active sides so that’s why optimizing the the pH the temperature during a fermentation process or any biochemical process is very important because if you are not able to maintain the conditions that are required for a reaction to occur you are going to lose producing the product that of or your the product of Interest so that’s why because when we are working with proteins when we are working with enzymes Things become a bit more complex you want things to be very very rigid in terms of the the temperature range or the PH range but if you are able to engineer these components in such a way that they are able to uh take care of their self for a specific range of temperature or specific range of pH there’s no modification in their structure that would be an amazing opportunity because that will reduce the rigidity of the process so the process might vary a little bit up and down but you still get the product of your interest you’re getting the point what I’m trying to explain right if it was very rigid like if my enzyme is only possible is only active let’s say at a ph7 what if my pH reduces to 6.8 will it be active anymore it won’t but what if it it works between the pH of 6.5 and 7.5 it will be because it will not change itself it will not change its structure within that range it will change the structure if it goes out of those range that is what we want through engineering enzymes we we want it to be much more flexible this is my understanding of engineering enzymes if you read uh literature you might get something else but this is my understanding of it now the engineering enzyme approaches that we have here so you have your engineered enzymes let’s say which is 20x uh increase in the half lifee at 70° C so you are working with liases so you do the solvent optimization you do a substrate product tolerance of it you improve the stability of it you do a site directed mutagenesis and then you do a computational design of it this whole approach is part of your engineering enzyme or enzyme engineering we are using the same synthetic biology tools to make modifications towards enzymes if we go back I might change the the slides here let me go back very very very quickly when we were discussing about the volum biomolecular engineering the example that you see here is of uh new random mutations are introduced in the genes for selected enzymes the cycle begins so you have your DNA you do a random mutations in it so this is your directed mutagenesis like you’re doing a directed Evolution now all these random mutations might lead to formation of different enzymes like same enzymes with different characteristics right and then you would be able to identify these mutations now you keep on doing that and then you see the changed enzymes are tested those that are more efficient at catalyzing a specific desired chemical reaction are the one or the mutations that are involved in that so what you have done is like you have it’s not just one DNA that you made the change you have multiple DN right so for one you did this for another one you did this for one you did this you kept on making different types of changes and you let it grow they produce the enzymes with the modification now that modification you test it against what you wanted to use those enzymes for whichever one worked you go back and see which which mutation actually led to that you take that strain and and then you continue it’s a very simple process right if you if you see it from this perspective it’s a very simple process but it’s a very effective one it’s long but it’s also very effective okay so directed Evolution mostly for enzymes and proteins for your case study to you have to think about directed Evolution they I don’t want you to blame me later on that I didn’t help so I’m helping you out little bit by little bit so you have your case study one idea we will discuss about that I think I have already talked about that in synthetic biology case study 2 comes in engineering enzymes most of the times sorry not most of the times for this year now let’s get into the established techniques of engineering enzymes we have already discussed this this is your directed Evolution which we talked about the novo design is brand new design you take the sequence you design your own enzymes based on what you want it to work with this is costly but sometimes important when you’re trying to develop drugs specific drugs it’s a very costly process but it’s an important process but you have to do multiple validations it has to be go through so you have your active side modeling you change the sequences of uh the enzymes itself and then uh you have the same irrational mutagenesis uh where your random and side directed mutagenesis are conducted uh you have your sequence of interest in there and then rational mutagenesis and then you have direct Evolution this is in my view the most common one and the most effective one the directed Evolution now entic degradation of various types of plastics uh this is an uh a paper or the work done by Cho at all in 2024 this is a recent paper I will show you the reference or you have the reference list in this particular slide deck at the very end so if you get a chance do download the paper and read about it if you’re interested in microplastics so different micros will generate enzymes which would be breaking these microplastics now the point is can I change the type of enzymes a particular microbe will produce by changing the feed stock that it is feeding on it’s just a question I’m not I’m honestly even I am thinking about the answer right now while I’m asking the question but is it possible do you think it is possible to change the type or to generate a specific enzyme by changing the feed stock because we under in my understanding not the whole DNA is expressed at a given time depending upon what sort of feed stock I am having my DNA might get expressed and that expressed DNA might develop a different type of enzyme time do you agree with this idea you will see some microbes thriving with this change in the source the carbon source that they get for their growth and that’s how they are capable of working or digesting the microplastics they’re digesting the Plastics these things these are some some other sequence in the DNA that gets activated this is my understanding of it which was hidden which was not active earlier but suddenly it got activated because the source of the carbon that they had is not there anymore so they have to somehow adapt their whole biochemical Machinery to take into account this carbon source which they can eat and survive and mutations move faster right so if one pro was not able to survive some mutation would have taken place for the survival of the second one the third one and the fourth one the fifth one and keep on going that mutation will keep on going and then you’ll come up you’ll end up with a a a strain that would be capable of digesting microplastics completely so that research is very very fascinating on microplastics so now this uh micro orms are inhibiting sorry inhabiting various surfaces of microplastics and they release various types of extracellular enzymes so you can see the list of enzymes here uh which are able to degrade these microplastics into smaller fragments so petas is the one which is going to digest the pet and then you have uh the lipases the proteases uh laccases cutinases so there are a lot of enzymes which are produced from different types of uh uh the materials so for the plas PSS for the pets these are the enzymes so which one can work on which type of biopolymer now enzyme mediated microplastic degradation would also involve protein engineering strategies which are employed in the modification of enzymes that can help intergradation of plastics so if you’re able to make modifications to enzymes in such a way that you can develop it that uh develop the active sites that can digest the microplastics that would be one way of interacting with uh enzyme engineering now comes uh the idea of synthetic biology the systems biology and the en engineering it’s a very interdisiplinary area just like your biotechnology it’s a very interdisiplinary area so understanding of system biology synthetic biology uh some of the computational aspect where you’re using python which can use into bioinformatics using blast fasta uh and learning the DNA sequence Technologies all of this will give you the main which encompasses everything this is why I said understanding synthetic biology will help you pretty much understand enzymes enzyme engineering and systems biology now it comes the systems biology part so holistic and dynamic interactions within the biological systems is required so the whole is greater than the sum of its part what it means is that understanding the whole of an equal let’s say I just I don’t want to understand just pathway of the equalize I would like to study the whole metabolic pathway of the equalize what are the pathways different Pathways that are there and how they are mapped if I know how it is mapped I would be able to engineer it in a much better way for my own benefit it’s like uh Jack of all trade master of none but you really want to become master of one in this case so you want to understand everything you don’t want want to be like just little bit of this pathway that pathway you want to understand the whole map of equal life how each and every pathways are interrelated what product comes out what product doesn’t come out and once you have that information you’ll be able to engineer it for your own usage so that comes under system biology so system biology is an interdisiplinary science that studies the complex interactions and the collection Collective behavior of a cell or an organism so your Genet genes will basically give you your molecular networks the molecular networks give out your cellular networks and the cellular networks gives out your organ networks now genes will lead to phenotypes right but those phenotypes that whole process to reach that phenotypic effect you have a metabolic pathway in between right and it’s not just one gene that is involved in that particular phenotype there are multiple genes that would be involved into giving that specific phenotype to you so understanding the interaction between those gen understanding the interaction between the metabolic pathice of those genes that led to that phen uh phenotypic change is part of the systems biology so this would be an example so you have an existing drug so let’s say if there is a patient uh that patient requires that drug to get rid of a specific disease so you give that drug to the patient whether and then you get a thumbs up or thumbs down whether it worked or it doesn’t work right so that’s a straightforward way of doing it but before any drug is launched we always try to understand its efficacy you remember the term that we I used last yesterday right the the doo 650 why 650 why 650 Mig because that 650 mg is more than enough to reduce your fever that’s why we cannot say how many glasses of orange juice you have to drink to be cancer free because we don’t know the dosage value so dosage is very uh is very easy to give for the chemicals because we can develop that efficacy so but that comes out only from clinical trials that dosage value comes out from the clinical trials even your covid-19 vaccines the one which we got in India I think that was astrogenic covid Shield that was developed by astrogenic that or astrogenic we call them same thing and uh rest of the world it was fiser and Monna yeah so Monna and fiser they were mRNA sorry M mRNA based right now this mRNA base that was an amazing application of synthetic biology in developing a vaccine now think about it just for a moment just to develop a single vaccine you have heard about flu shots like people get a flu shot or flu vaccine like in North America we always get like every flu season there’s a vaccine but that particular vaccine is good for the flu or the whatever the the host was last year not this year so I got the flu sh before I came to India but I got the flu sh for the flu that was last year this year one comes next year so it’s it’s a long process because they have to sequence it identify develop the vaccine and then only then you get it right but how was this covid vaccine produced so quickly it happened and within a few months the vaccine was available that’s why we were able to save so many people right it was all because of the understanding that we have had through systems biology the Sy synthetic biology because once we knew how the whole Pathways work we were able to do that genetic circuits create take out the sequence which was impacting the active sites related develop the vaccine and make it ready for the market it was so quick but it took a lot of time but you do need that clinical trial aspect to it right that is where existing drugs come into F so where you have to go for the clinical trial so you have your molecular biology you do the clinical trial and that you develop mathematical model using the system biology thing and then you can have this personalized drug which would work for the patient because not everything will work for the patient I’ll give you an example to it so rosasa a rosu or R it’s much more easier so R which is a cholesterol lowering drug its interaction is consistent it’s it’s like it’s a very consistent drug from one human to another human it does not it has nothing to do with your uh what is that called variabilities so like the model helped researchers understand why the drug Works consistently despite genetic variability it will keep on changing now it was mostly physiology like you have these two terms like pharmacokinetics and phod dnamic model both of these models were used to develop this particular drug so how the like once you take the drug how does it travel within your body that was understood what is the site at which it will go and work that was understood once it reached the site what does it do that was understood UD once you have all of that information you can develop a drug that would be consistent for me consistent for you consistent for the other it will work in every human person with the same efficacy that understanding comes from understanding of the systems biology I don’t want you to like remember this I’m just giving this as an example this is an application of systems biology the other comes the omix and the Cascades of the omix for understanding your system biology because you need to understand each and every metabolic pathways and the proteins that are being generated by each and every Gene the rnas you have to go for epigenomics you have to go for genomics transcriptomics proteomics metabolomics cellomics and the single cellomics once you have all of this information you can make maps or networks so you’ll have this particular so let’s say this is a genetic sequence this is giving this this is giving to another Pathways this will lead to other set of reactions and so on so you will be able to make a genetic map or metabolic map of the whole sequence and this particular casket is basically your flow of information through the biological systems it gives you the hierarchy of how the things go through so you have your hones modification DNA methylations 3D organization of Chromatin that goes into DNA then DNA gives you RNA RNA gives protein protein gives you metabolize metabolize gives you cell functions and the phenotypes and then you’re getting back so synthetic biology and systems biology have a synergistic relationship so system biology is understanding integration and prediction using computational models so once you have all that information using all the omix you can develop mathematical models synthetic biology is control design and Engineering using computation model you’re designing new things specific uh molecular events so you combine both of them you get where the system and the synthetic biologies are used in Synergy to have a desired system Behavior so if you want your system to act in a special way or specific way use both systems biology and synthetic biology understanding of both the sector and then you’ll be able to get uh what you want most commonly for Therapeutics and Drug Discovery it is very necessary that you have these understandings so if you have a background in molecular biology if you have interest in molecular biology cellular biology develop more of that interest in computational biology look into systems biology look into Synn biology use that and buy informatics for sure for drug Discovery so these are the references the Choy at all should be somewhere here if not I’ll will find it yeah so this one is the paper so recent advances in microb enzy entic engineering for biodegradation of micro and nanop Plastics so this is one of the papers that my student was following for his research and he helped me develop the slides for for this particular session we’ll take a break for 15 minutes and then we’ll get back for your case studies we’ll have a brief discussion about it because we have a complete session on this case study 1 and case study 2 later but I want to I want you to understand what they are what we are asking for and it will also require you to work in groups if you can divide yourself into groups depending upon your own comfort zone I am I’m not aware who is comfortable with whom so if you can divide yourself into groups that would be easier because you would be working let’s say on like sort of like a term paper at the very end but it’s not I’m not asking you to write me a 50 page or 60 page term paper I would like you to develop the whole process for me so if this is the product that I want this was the approach that I followed so I first went here identified the sequence I did this this is the host that I selected why I selected what was a criteria that was based on it what I did after that and how I did it and what sort of fermentation system will I choose to grow this particular product and what would be the process proc parameters so you might have to do some Google searches but it would be an interesting experience because this is exactly how we develop research projects as researchers we only know the product now we have to develop the whole experimentation so cool so we’ll take a 15 minutes break and then we get back here welcome back after the second break now for today we have had enough of uh the theory aspect uh what I would like you to do now is relax a little bit think about your case study the case study one the case study two these two are the most interesting part of the whole Gan session in my viewpoint uh what I would like you to do and I think I have actually had ask this on the very first day that you do need to divide yourself into groups so for the case study one case study 2 on the very last day that would be the Friday we will be you’ll be presenting a couple of slides from each and every group so if you divide yourself into four groups or five groups depending on that you’ll be presenting a slide on your case study one for the case study one session and the on case study 2 for the case study 2 session and we’ll have a conversation an interaction you will be questioned by your colleagues and you’ll also be questioned by me why you chose a specific host why do you choose a specific reactor design why did you choose these parameters what was the intent if you’re choosing 10° Cel what is the reasoning for 10° celus you should have answers to those type of questions and if there is something missing I will try my best to answer those part of the questions for you okay and that that would be a very interactive session so my humble request is do divide yourself into groups and uh give your group a fancy name not something that would be hurtful to others but something that will add value like something that you will be interested in to like let’s call your yourself as a startup in that case so give that a startup name and then we can go ahead from there okay but this part of the session I’m just going to give you a brief idea of what I’m expecting for your case study 1 and case study 2 okay so the case study one was developing a plant protein so the protein that I wanted or I want you to develop is monoline now monoline is a single Alpha Helix packed against five an sorry anti parallel beta strands in a bet graph fold 16 fold is500 the most important part of the modeling is 1 1500 so three 3,000 times sweeter than sucrose say it’s an alternative to Sugar not like Stevia better than Stevia it’s a protein okay it’s a plant protein I would like you to develop a process one which you are seeing there is the one which I would be I would be using so the host microorganism that I would have chosen would be uh just that’s a yeast I have the amino acid sequence I have not provided you a DNA sequence you need to tell me what would be the sequence for your plasmid or whatever uh process you’re using for inserting your DNA into any host and then how are you going to transfer it into to your reactor what would be the feed stock in the reactor do you need any growth factors or nutrients what would be those nutrients you would be able to identify or find those information online on Google you don’t have to go through uh a tons of research articles even if you do a normal regular Google search you would be able to identify enough information to answer that question okay and then once you get your product whether it is intracellular or extracellular depending upon the host you choose and what would be the purification and downstream processing steps that you will choose and to add little complexity to it I would like you to perform a simple tea which is your technoeconomic analysis in a technoeconomic analysis you look at what would be the cost of your raw material what would be the cost of the handling of the raw material uh whether the technology exist if it is available ready available in the market so let’s say if you’re looking at sonicator for purification or extraction of uh your intracellular uh protein then bless you if you’re looking at intercellular protein you you are going to use sonication to do the cell lices what is the cost of a sonicator you identify that on Google I’m not asking you to give me the perfect value to it but I’m give I’m asking you to give me an idea how you will approach because the problem is what I want want you to understand is that when we develop a particular product it’s not just the value of the product depends on the process itself so if I’m if I have a very costly process but I also have a super costly or super valuable product I would love to invest because I’m still making money right so if if modelin is going to replace the whole sugar industry in the food industry if I’m going to capture the whole food industry why will I not invest even if the the the process is costly I’ll do that so that technoeconomic analysis that’s why I want you to work in groups because then you can divide the different stages of the production between yourself and then you can identify the economic analysis for those sections and then you are going to present it to us on the very last day as part of the session okay and then we can have a very good conversation why your product costs more and their product cost less and we can look at you can have a competition who is going to sell me model for cheaper prices okay so the benefits of monoline is the Dig has no impact on the blood sugar level that’s why I’m very very interested in it but this is the biggest constraint a heat treatment over 50° C at low PH denatures monoline question for you then how will I use it in food industry because the products if I let’s say if I’m adding sugar to a juice I’m going to add it before the pasteurization or after the pasturization if I have to maintain it the structure what if I add before if I’m adding it as a natural sweetener at home and then as I’m making tea I add sugar before as the tea is boiling I’m adding the sugar to it right so the the protein will denature what will you do to pre uh prevent that think about it okay so that can be a part of your solution people have actually answered this in the in the during the day session how will you preserve something which is thermally sensitive you just take a starge that’s the max hint I can give and then you do whatever you want okay the second case stud study that we are going to discuss on Friday is developing a microbial protein now here we are going to work with Alpha mileage so you’re going to produce Alpha mileage which is an enzyme so all enzymes are proteins but not all proteins are enzymes so I’m taking the benefit of that and giving you an enzyme how will you get Alpha milees so there is a process that I would use if you are going to use the same process I don’t mind if all of your all the groups are going to use the same process I won’t mind you can change it if you would like to uh but I would love to know the host that you are going to use to grow these for the fermentation process and what would be the purification process that you will follow for that okay Alpha Ames in bacteria used for breakdown of starch as source of energy it is used in production of food detergents bioethanol and paper the if you remember the example that I gave you when the cats pee in specific area you have the urine a mileage is is ALS is used for degradation purpose because it acts as a detergent too right for the cleaning and removal of the aroma so in bread for reducing hardness uh enhancing elasticity and delaying stalling and for the gene modification you can the gene modification would be required to get certain properties of to the enzymes right we do directed Evolution because we want to change the property of the enzymes we want to make it much more stable we want to make it viable at uh higher temperature ranges or pH ranges right so depending upon what sort of process you will choose you would be affecting the property of your enzyme I’m choosing Alpha amyes because that’s a very common enzyme and most of even if you are from the background of food you will have some understanding of alpha mileage okay so use that and I wish you all the luck for that uh to work in a group and come up with the solutions so do you think these two case studies are good enough for you or you want another one two or more more than enough okay so I’ll keep it at two and uh some of the tools that you can use for yourself would be your the blast if in case you are trying to identify the sequence for monoline I have only if you have taken a picture of my slide you would only get the sequence of the amino acid you’re not getting the sequence of the DNA you have to find the sequence of the DNA okay so take as many pictures as you want and if I’m there in that picture I would be I will not mind that but anyways uh so blast faster you can think about brushing up your B informatic skills a little bit if not you can look into YouTube how to do certain things or if you can take help make your group in such a way that somebody knows what to do okay you can use the pdb database Bank all proteins are sorry all enzymes are proteins but not other way around so if you want to know more about Alpha mileage where will you go pdb okay if you want to know more about modelin you will go pdb so use these tools and databases to gain more insight you’ll also be able to find the source who would have worked first on monin you can read those research articles those are freely available or you can look into those research articles how they got it okay that would be it thank you for the session today