hello champions and welcome back to pw english and i am your dikshama so today we are going to study the chapter biotechnology principle and processes so from any point of view if i'm talking about cbsc i'm talking about neet exam this chapter is really very important also you will get to know or you will get a glimpse of another field of science that's biotechnology and it is one of the emerging field in the science so if i talk about biotechnology let's just don't discuss about the questions and all i'm talking about the general thing about your life in biotechnology uh if you really don't want to be a doctor you have no interest in becoming a doctor but you have still medical you are in a medical field so this is one of a very interesting field you can opt for later in life or there so there must be a question in your mind how can you enter in this stream so you can directly enter into this stream after 12th class after 12th standard you can give some entrance examination of universities where you can get this degree of bse by technology or there is also another degree in the b tech if you have mathematics with you you can also get into the engineering field of biotechnology as one right so it's kind of my emerging field as you know today the vaccination that has that has saved millions of people on this earth against covet right or it has produced immunity against covet this is just because of biotechnology so it's like a boom as well as a gift to us to the humanity so let's uh talk about it let's discuss some of the topics or the in fact the entire chapter i say of biotechnology so first of all what is a biotechnology so if i talk about it there is a federation european federation of biotechnology they have given a definition of biotechnology uh that is the integration of natural science and organisms sells parts thereof and molecular analogs for products and services so what we do in a biotechnology if you see this term if you see or realize that this term biotechnology have two terms in it one is biology another is technology that means you are you know associating two fields one the biology another the engineering field together and that becomes biotechnology so definitely you're going to engineer something you are cutting and making new things this is what an engineer does right so uh you are using natural organisms you're using natural science you're using biology you're using organisms they're cells you can also use their cells or the entire organism you can use entire organism you can use their cells or any parts as well or or the molecular analogs now what are these molecular analogues for example i say one organism or cell is producing x right this is what it naturally produces by learning its chemical composition i produce the similar product i used or i produced a similar product in my laboratory and that's molecular analog for example insulin is also produced in a body and insulin you also produce it in your laboratory so that's an analog okay or there are certain type of things that looks like insulin they are known as analogues or like there are certain hormones that looks like your natural hormones in the body for example progesterone like or the analog is lng20 so this is how you can either use the entire organism or you can use the cells parts or the molecular analogues for your products and services for example what's a service say you want to give insulin to the diabetes person right diabetic people so that's the kind of service you're providing or you're giving to the person so this is the entire definition of biotechnology given by the european federation so if i talk about biotechnology biotechnology have biotechnology have two fields or i say the biotechnologies of two type one is the traditional biotechnology and another is a modern biotechnology okay so uh the biotechnology term has it's not like uh you know very old term it's a recent term reason doesn't mean it has this game like few years back but yes many years back but as comparison to the other fields it is a very new field but it doesn't mean we were not using bad technology later then we were using the biotechnology but the name was not given for example your mom produces curd at home that's about technology for example people used to make dosa right in dosa we use fermented batter that's about technology we use fermented batter for uttapam as well so all these food we use fermented uh this uh you know wheat or bread we've produced bread fermented bread right like bature in the chole that's also fermented so all these things they come under the traditional biotechnology whereas a modern biotechnology is you know a little step ahead little step ahead than the traditional biotechnology how so you are producing the um curd at home fine and this curd is also produced in the factories at a larger scale where they you know they keep in mind what type of bacteria they should use they keep in mind whether the bacteria is in acidic culture or the alkaline culture they're taking care of all the quality and other things that becomes modern biotechnology for example people also make alcohol at home that's traditional like toady right they are also producing in the big barrels in the industries taking care of all the things so this is how the traditional and modern biotechnology is different from each other but since they're doing the same thing they're performing same per function so they are the part of biotechnology only right okay so let's move further and talk about the principle of biotechnology so there there are two things in this chapter if you have seen the topic of the chapter what was it the principles and processes okay the principle tells us the techniques we use and processes tells us how are we using these techniques okay so there is only two things in this chapter first what are the techniques and second how you do it for example whenever you perform an experiment at a school or wherever like college is then all first of all the teacher will give you a demonstration uh about the experiment you're performing okay so the teacher will tell you how the experiment is done they will tell you about all the reason behind it all the science behind it everything they'll tell the theory of it okay but when you go and use the equipments and you perform it and you produce something you produce the result that's what you are doing so these are the processes so first we'll discuss about principles what are the techniques used and then we will see how the techniques are used so if i talk about the principles used there are two basic principles of biotechnology there are two basic principles of biotechnology one is the genetic engineering one is the genetic engineering and the second one is maintenance of sterile culture maintenance of sterile culture okay so this is how you sum up the principle so this is how you sum up what is done in the biotechnology what is genetic engineering genetic means gene engineering means you are cutting something you are joining something so in genetic engineering we cut the dna we join the dna we form the recombinant dna we form the recombinant dna all right and then we also introduce a dna into host we then introduce the dna introduce dna in host and then we produce products and then we produce products okay so this is what we do in genetic engineering i will explain you this entire process in in detail in the later sections okay whereas in maintenance of sterile culture so this is the basic experiment you are doing but while doing this experiment you need to take care of one very important thing and what is that you need to maintain sterile culture where what is sterile that means contamination free that is contamination free for example if i'm growing a culture of bacteria and the culture of bacteria is grown in agar you must have heard of it right so agar uh you're you're producing or you are you know growing the bacteria in agar but agar is also favorite food of fungus do you think the fungus will not come on that favorite food definitely it will but if you do the experiment to perform the experiment in the sterile conditions for example in a room which has no contamination there will be no fungus in the air it will not enter into your culture culture is what you are growing and there will be only bacteria that will be growing in the later form but if you do you do you you are doing something like that your hands are not lean or your air or the room is not clean where you're performing experiment the culture will catch the another microbes like fungus and that will cause contamination so you have to make sure whatever you're doing there it should be contamination free okay so first thing contamination free equipments you should be contamination free your hands your mouth should be your face should be masked and everything like that so you have to take care of everything and second this is the experiment what you do so now what you do in in the genetic engineering so this is one of a basic thing that you do gene cloning so i am performing one experiment that is gene cloning i am multiplying a gene by using genetic engineering so how i will do for example i have one this beta cell this is beta cell of human right and this cell is like any other cell with dna with dna double standard dna right yes so what i'm doing here is i want one fragment of this dna and that's a gene what gene i'm taking i'm taking gene that is producing insulin okay so i want i want my bacteria to i want my bacteria to produce insulin so how does it happen or i want this gene to multiply i want this gene to multiply and i want a lot of copies of this gene how will i be doing this first of all i will break the dna and i will isolate this gene by using the enzymes that is restriction enzyme which enzyme restriction enzyme restriction enzymes are certain enzymes which are molecular scissors they are like scissors for example you have a ribbon so how will you cut the ribbon you need a scissor just like that that ribbon is your dna and you want to cut it with a scissor and these pair of scissor are your restriction enzymes okay similarly i want someone i want someone to take this gene to take this gene and put this gene put this gene in the e coli what is my purpose here i want the bacteria e coli to produce insulin so for that i have isolated the gene insulin gene from my cell and now i want this gene to enter into e coli this gene is not capable to enter into the e coli so i need some transporter i need some transporter which can easily enter into it and i will give that gene to that transporter you are going so please take my gene as well just like that okay so who is this transporter these are vectors these are vehicles you know what are vehicles like car your bus everything like that so i'm also cutting the vector somewhere because i want this vector to take this gene so somewhere i have to fix it so for fixing it i will also cut this with restriction enzyme now this has a cut now this has a cut it can easily accommodate it it can easily accommodate it now it will enter into the e coli it will enter into the e coli so see this has entered into the e coli so when it has taken my gene so for this this gene is insert or foreign you also call it as insert so now this dna has become recombinant dna what is it it is recombinant dna so now this recombinant dna is entering into the e coli so i will call this process as transformation i will call this process as transformation so there are a whole lot of things so do not get confused it's quite simple just prepare your mind that you're performing an experiment okay so what i did i i took my cell i have uh isolated one gene of interest you also call it as in insert then i inserted this into some vector now vector is going inside the e coli so while it is going it will also take your gene of interest or your insert so the process where this recombinant dna because now it has combinations it has two things so you call it as a combination so this dna is recombinant dna now now this recombinant dna is entering into the e coli so this process very common in dna is entering into the e coli is known as transformation what do you call it as you call it as transformation process okay now while e coli is multiplying it will also let this vector multiply as a result the number of gene copies will be formed so now it will multiply equalize multiplying while it is multiplying it will also produce a lot of copies of this recombinant dna along with it and as a result your gene has been produced so this is what is known as gene cloning clone means producing the exact copies what is a cloning loaning means producing exact copies exact copies okay so we have produced a number of copies of these gene by just entering into the e coli all right so this was about the gene cloning so this first recombinant dna was produced by someone this must be produced by someone so who was that person who uh for the first time has done this experiment that was herbert boyer herbert boyer and stanley kohan so these were two scientists in 1972 they performed one experiment now what was this experiment they took a bacteria that salmonella salmonella okay this salmonella this salmonella has this vector and that's plasmid you must have heard of a word plasmid before these are extra chromosomal strands of dna in the bacterias yes so this is the plasmid of the salmonella and since this bacteria is antibiotic resistant this is antibiotic resistant so it has a gene it has a gene that makes it antibiotic resistance so this gene is antibiotic resistant gene antibiotic resistant gene okay so you understand what is antibiotic resistance for example i have this salmonella it has this gene antibiotic resistance for example i have antibiotic x an antibiotic x if it is given to salmonella the salmonella because it is antibiotic resistant it will not die antibiotics are certain drugs which you when you give to the bacteria bacterias they die which are sensitive to antibiotic but the one which are resistant to antibiotics they will not be uh or the antibiotics will not be able to kill them so they can easily flourish and live happily even in the presence of antibiotics so what these scientists they does they isolated this gene by how they will isolate it by cutting it so for cutting it you need restriction enzyme so they isolated the gene with the help of restriction enzyme so they isolated the gene so this is antibiotic resistant gene so they isolated antibiotic resistant gene resistant okay so they they isolated antibiotic resistance gene with the help of restriction enzyme from salmonella and then they added they added this into they added this into a vector they added this into another plasmid and this plasmid was antibiotic sensitive this was antibiotic sensitive so this is anti biotic sensitive so they added they added this gene they added this gene into this so what was made what was made a recombinant dna was made a recombinant dna was made so this plasmid is now having antibiotic resistant gene this plasmid has antibiotic resistant gene so wherever it will go that bacteria will become antibiotic resistant so they took a bacteria they took a bacteria and this bacteria is antibiotic sensitive this bacteria is antibiotic sensitive now what they did they they entered this recombinant dna which is basically a plasmid which has the gene of antibiotic resistant bacteria is this this dna recombinant dna is now entered or it is now entering into this into this antibiotic sensitive one so i'm putting here so now this recombinant dna which have antibiotic resistance gene it is going to enter into this now this will become antibiotic now this will become antibiotic resistant now this will become antibiotic resistant okay so this is how it is anti biotic resistant okay so this is how they have made the first recombinant dna so the first restriction enzyme to be used was their hind2 what was it hint to so we'll be discussing this in the later section so don't worry about it so uh this is how in 1972 these two scientists they performed this experiment for the first time and they made the recombinant dna all right moving further to the next what are these so what do you think what are the basic step that one should keep in mind performing these experiments first the identification of dna with desirable genes so first basic step first basic step you have to identify which gene you want to extract second then introduction of dna into the host so after that the second crucial step is that dna should definitely the recombinant that you have produced that should definitely be entering into the host because if it doesn't enter into the host it will then be not be able to multiply maintenance of introduced dna in the host and transfer of the dna to the progeny now if it has entered now if it has entered into the host that's another crucial step now even if it is entered whether it is going to multiply or not whether if it is going to multiply it is going into the next generation or not so these are the three different crucial steps that you need to take care of while doing the bio technology okay moving further moving further to the next that is tools of recombinant dna technology so now what are the tools that you use okay so performing the experiment for definitely that you need something for example if i'm performing the experiment of chemistry i am mixing two chemicals so what is the two basic important things i need there two chemicals just like that we also have certain tools here the first tool that i have told you here what is that first rule that i have told you is restriction enzyme okay second tool you need to have this vector that's why it is going to entering somewhere and that is the host so if you have these three things you can easily perform this gene cloning so what are these three things of recombinant dna technology so what are the tools of recombinant dna technology so why first of all why we are calling it as recombinant dna technology because we are recombinating the t dna you're producing combination of the dna so we have basic three tools first of all as you have seen we need restriction enzyme so we need some set of enzyme it's not just restriction enzyme we need some other enzymes as well so first is enzymes second we need the vectors vectors are vehicles that will allow our insert to enter into the third tool what is it the host so these are three basic things that you need now what are that various enzyme first of all you already know what we have introduced restriction enzymes one type of enzyme that you know are scissors second are joiners these are ligase these are glues they will you know join both the ends of dna and then we have alkaline phosphatase phosphatase and then we have dna polymerase so these are the enzymes that you need for product for performing these experiments okay so we'll start with the first one that's restriction enzyme okay so water restriction enzyme as i've told you restriction enzymes are molecular scissors these are molecular scissors so from where do you isolate this or who produces these enzyme who produces these enzymes these are naturally produced by bacterias for what for protecting themselves against the viruses so these are produced by bacterias these are naturally produced by bacterias naturally produced by bacterias produced by bacteria okay so right now we have isolated around 900 restriction enzyme from over 230 strains of bacteria so the this is a number that still we have so what's the discovery behind this let's talk about how they were formed okay so there was a group of scientists who were performing some experiments how the bacteriophages the viruses they infect the e coli how the bacteriophages infects the e coli so while they were performing experiment they i'm writing here about discovery so what they were doing is so they were performing some experiment so this is e coli this is e coli so on e coli one virus which is bacteriophage i'm producing it like this okay so you know how does it looks like so bacteriophage you know already how it looks like i'm just making a cartoon of it okay so you have seen that in botany only so they were saying how the bacteriophage infects the e coli so in turn what does e coli does so e coli produces two types of enzymes here one are methylases another are restriction enzyme okay so why the e coli was producing it so virus have one thing whenever they infect a bacteria they will give their dna to it they will give the dna and they will hijack the equalized dna okay so what does e coli do it will produce methylase enzyme this will me this will add methyl group this will add methyl group to equalize dna okay so these enzymes are produced by e coli only so one enzyme will add the methyl group to the entire equalized dna so now e coli knows whatever dna is methylated that's mine and whatever dna is not methylated that's virus dna or viral dna so now it will produce a second enzyme that's restriction enzyme it will cut the non-methylated or unmethylated dna and you know the non-methylated dna is of virus so this is how the e coli is protecting itself by producing set of enzymes from the viruses or the bacteriophages okay so this was how the restriction enzyme thing was introduced and they get to know yeah there are certain enzymes they can cut and chop the dna but the after like around five years of this thing they isolated the first restriction enzyme so the first isolated restriction enzyme was in the two it was hint two okay after around five years of this discovery it was isolated isolated means you segregated it you know there is enzyme in that mixture and it is cutting the dna but then you isolate it you you you know you put that out and you see what does it looks like and how it cuts it and this was done after five years of this discovery so basically since they're molecular scissors so what did what do they do this is a molecular scissors so they're chemically type 2 endonucleases type 2 endonucleases what is type 2 endonucleases endo means within within nucleuses means any enzyme that cut dna so the enzyme that cuts the dna they are nucleuses they are of two category one is exonucleases another is endo sensor restriction enzyme cuts the dna within so any set of enzyme that cuts the dna within not from the site they are the endonucleases when do nucleuses further have different categories so they belong to type 2 category which category type 2 category so the function is to chop the dna the function is to chop the dna now that's okay they are enzymes because they are chopping the dna so which bond they do they break for the dna they break the phosphodiester bond you know the dna is backbone the backbone of dna is made by sugar phosphate yes it is made up of sugar phosphate so it is technically attacking there only it is technically attacking to this spawn only okay all right so since they're endonucleases you also call them as restriction enzymes so why the word restriction is used so whenever so whenever whenever the restriction enzyme works what does it do first of all we have this uh dna strand okay we have this dna strand first of all it will recognize a sequence gaa ttc for example this is the sequence of uh your dnas right this is the sequence of dna and this is a particular gene this is a particular gene okay so first of all what does this restriction enzyme do it will you know trace the entire dna it will trace the entire dna it will read the dna sequence for example i have one restriction enzyme eco-r1 eco r1 will recognize a particular sequence of dna echo r1 will recognize a particular sequence of dna and that's this one gaa ttc ego r1 if it is given by any other sequence it will not be able to recognize it so this is how we say this is the recognition sequence what do you see you say this is a recognition sequence so every restriction enzyme this is equal r1 it's a restriction enzyme this is one example every like other they have their particular recognition sequences so first of all the enzyme will read the recognition sequence and the sequences of around four to eight nucleotide long how many four to eight recur four to eight nucleotide long after it got to know yeah that's my recognition sequence now what it will do now it will sit on a restriction site now what's a restriction site restriction site is a particular it's a particular nucleotides restriction site is a particular nucleotide where it will give a cut okay for example i have that ribbon now where i'm going to cut it there is a particular site before this or after this okay so there is a particular site and that you call it as restriction site so the restriction site is somewhere here so this is the restriction site so this is you call it as the restriction site where it is going to cut so because it is cutting at a particular site you it is restricted to a particular site so that's why they got its name restriction endonucleases or restriction enzyme so when it is going to cut here now what is going to happen next when it will give a cut it will give a nick here this is going to form one sequence because it is going to cut in a way like this okay because once the phosphodiesters bonds are broken the hydrogen bonds here it is they are very weak bonds as comparison to phosphodiester they are definitely going to move apart okay so when they give a cut the entire sequence will move in that manner okay so one sequence will get this one of sequences another sequence of dna will be formed like this a a t t c and here g okay so this is what uh the the or this is how the dna sequence is going to get broken into two pieces so if you see here if you see here so these type of ends are formed if you see these type of ends are formed so you call them as sticky ends what do you call them as these type of ends are known as sticky ends what do you call them as sticky ends so here there are two types of ends are formed depending upon the restriction side depending upon what the restriction side for example we have taken the example of equal r1 okay for example if i say i have another restriction enzyme that is sma1 sma1 has a sequence of this c c c g g g so g g g c c c this is the recognition sequence it recognizes and the restriction site is somewhere here so when it is going to broken down what it is going to produce it is going to produce this so as you can see the ends are blunt here ends up blunt they are smooth ends so you call these type of end as blunt ends these are blunt ends so depending upon what type of restriction enzyme you're using and what type of you know restriction site they have two types of ends are formed one are the blunt ends and another are the sticky ends sticky ends are also known as cohesiveness why cohesive cohesive means that can easily be stuck into that can easily be joined so because if it has this type of end the blunt ends they will not be able to stick easily why because first of all you need to form the phosphodiester bond only then they will join okay but here but here these if they are like brought together near to each other the hydrogen bonds will be formed between them and later on there is more easier to form phosphodiester bond okay so that's why the sticky ends they are more easier to get together or they can easily join it okay so this is how the restriction enzyme works by recognizing the recognition sequence and by cutting at a particular site you can get two types of end depending upon what type of restriction enzyme you are using so now one more thing that you must have noticed one more thing you have noticed their names their nomenclature so how do you name them you must have noticed two things the first thing is nomenclature so for example if i have this eco r1 how do or how does it get its name e means esthersia azurisia okay co means coli so that's the genus that's a species okay species and genus and r is the strain r is the strain whereas one is order of discovery one is order of discovery for example we have equal r two also so the one was the first one to be there or one was the first one to be discovered whereas two was discovered later on okay all right second thing you must have noticed are the palindromic sequences restriction enzyme always cut they always cut on palindromic sequences what are parandomic sequences there are sequences that repeats itself for example if i have g a a t t c so how does it repeat sir the same way g a a t d c which is going reverse it is going reverse yes yes or no so we have c t d a g g c t d a g so it's repeating itself so these type of sequences are palindromic sequence and restriction enzymes always they always cut on palindromic sequences and which bond do they break breaking phosphodiester bond breaking phosphodiester bond okay so this is how the restriction enzyme you have a lot of restriction enzymes and this is how they perform very very very crucial functions in your uh system right so not their present not in your system but in the biotechnology where are they producing they produced naturally by the prokaryotes which prokaryotes bacterias so that they can fight against the viruses so this is how you have isolated them and now you're using it for your own purposes all right so that's about the restriction enzyme let's talk about the another enzymes what are those then we have ligases dna ligase ligase ligase are molecular glue they are like fevicol they're like fevicol okay so fevicol was a function of fevicol if if anything has been broken down into two pieces you're going to stick them up so what uh what is uh what have you done you have made two ends of dna right so these two ends for example you have produced these two ends you need to join them together so for these you need dna ligase and the dna ligase we use here is t4 like is t4 is the virus from where you have isolated them okay so from there it has been isolated and this is used to join them so that's why it's a molecular glue so how do it join them i've told you you need to produce phosphodiester bond so restriction enzymes are cutting the phosphodiester bond whereas dna ligases joining it by producing the force of drastic bond so these enzyme they produce phosphodiester bond between two nucleotides okay because dna's backbone is made up of sugar phosphate okay so that's a nucleotide it's uh if i say the monomer of protein is amino acid the monomer of sugar is monosaccharide just like that the monomers of dna is nucleotides so we have a sequence of nucleotides all right okay so the next enzyme that we have discussed is dna polymerase you all know what does dna polymerase does because you need to multiply the gene you want dna to get multiplied so dna polymerase is used to replicate the dna replication of dna and the one enzyme you must have not heard of that's alkaline phosphatase alkaline phosphatase now what does it do it cleaves the phosphate group it cleaves the phosphate group what does it mean let's see for example i have one plasmid okay i have plasmid like this now i cleaved the plasmid here so what is produce a strand of dna this dna okay uh all right so let's make it in the same manner so after cleaving it this is formed for example this end is 5 prime and this end is 3 prime so forming a phosphodiester bond what do you need a five prime end and a three prime end only so can't they be able to join they can definitely join so so to you know uh you know to get these chances of rejoining so that they should not rejoin or to you know do not let this accident to happen what we do we cleave the phosphate here because five prime hand have a phosphate so we cleave the phosphate here from with the help of enzyme alkaline phosphatase so as a result the phosphate has been cleaved up here now this end will not be able to join with the three prime now why we're cleaving the phosphate because five prime end which have the free phosphate and three prime end which have the free oh they are going to form phosphodiester bond this is what we do in a biomolecules also okay so if i don't have phosphate here if i don't have phosphate here will it be will be able to form the bond no bond will be formed so that's what we do we use alkaline phosphorus phosphatase that will cleave the 5 prime phosphate from this end so that no joining or rejoining should occur so we cleave the phosphate group from which end from five prime end to prevent rejoining of dna okay all right so this was about the enzyme let's talk about vectors so what are vectors vectors are vehicles what are these these are vehicles to transport insert or gene of interest transport the gene of interest in host so definitely if it is going to become a vector it should have certain qualities should have certain qualities now what are these qualities that a vector should have let's see so the first quality is the first quality is it should have origin of replication origin of replication is very much important if it doesn't have this will be it able to multiply no it will not be able to multiply remember whenever you study dna replication you what do you study here you see the replication starts at alright or region of replication if a dna does not have will be able it will be able to replicate no and replication is very important here what are you doing you are doing gene cloning you need replication here so origin of replication is very much important here second thing is selectable marker second thing is selectable marker what are selectable markers selectable markers are usually antibiotic resistant genes these are antibiotic resistant genes like cannabis and resistance ampicillin resistant like tetracycline resistant genes or these can be certain genes of enzyme like lac z gene lag that produces beta galactosidase now why do you need these genes because for example we have done all the experiment you are not doing it under the microscope all the time you cannot see whether the vector is entering or not so to see whether it is entering or not whether the vector is entering into the host or not you need to have something that will tell you the or it is like an indicator that will tell you yes yes yes the vector is containing the gene and it is enter it has entered the host otherwise you will not be able to identify by the naked eye whether this contains bacteria and the bacterius contains a recombinant gene or not right so for that you need an indicator so these are the indicators indicators they will distinguish or they will distinguish between recombinant and non-recombinant whether the recombinant gene has produced or not so you must have these okay and third thing is mcs multiple cloning site now what are these multiple cloning site these are restriction sites these are restriction sites of various restriction enzyme because you want to cut the vector and you want another gene to get in so for cutting it you need that particular sequence for example i have a plasmid and i'm using eq r1 and you know for cutting eq r1 we need g80dttc sequence of dna but if the vector doesn't have that sequence will it be able to work there it will not be able to work there so for that you need those sequence of dna where the restriction site restriction enzyme will work these are what these are recognition sequences of various restriction enzyme so you need multiple cloning site what are these these are recognition sequences recognition sequences of various restriction enzyme various restriction enzyme so you need to have them you need to have them if you want to cut the dna and if you want another dna to enter into it to form recombinant dna okay all right so these are three things your vector must have so we'll start with the first vector that's a plasmid what are plasmids you must be knowing it they are extra chromosomal dna of the bacterias and yeast extra chromosomal dna that means the dna other than the main chromosomal dna okay where do you find them you find them in bacteria you find them in the yeast right so these uh chromosomal or ring-like structure these are circular dnas what are they they are circular sometimes they contain antibiotic resistance gene what do they contain antibiotic resistant genes with the with the various techniques or you can say with the advancement of biotechnology we have uh you know modified these plasmids and produce them according to our needs for example the first plasmid is pbr322 how does it get its name pbr322 p means plasmid okay let's focus here everyone yes see i know you guys must be feeling like oh my god what's happening oh it's bad technology but it's quite interesting go step by step it's quite interesting chapter so pbr322 got its name from p that's plasmid b and r are the scientists oliver and rodriguez broadray hughes and 322 is the order of discovery or you know it's like a roll number they get for example in the school you get the roll numbers this is how you get everything there your id and everything right so this is like the index number okay so this is how it looks like because it's a double standard so you can see two strands of dna here it's extra chromosomal and it's circular so you will see the antibiotic resistance gene first gene is tetramycin resistant here the pink one another is ampicillin resistant gene then you can also see your eye here you can also see the various restriction site for various restriction enzyme and then we have rop what is rop rop codes for the proteins involved in the replication of plasmid so where whatever proteins we need for replication there are different types of protein for winding and unwinding so these are coded by these rop okay that's a segment of gene or the dna okay so this is how it looks like so now how do you put the insert and how do you get to know whether uh this one is having the gene or not or or simply saying that how do you get to know its recombinant or non-recombinant it's transformative non-transformation let's see let's perform some experiments let's get started so now let's see how can you distinguish a recombinant or a non-recombinant and transform the non-transmitted so for that let's clear a basic software recombinant in non-recombinant bacteria and transformative non-transformation for example if i have one bacteria i have one bacteria and this bacteria is having a plasmid it is having a plasmid no matter what what type of plasmid it has it's a transformation what is it it's a transformation so if any of the bacteria has a plasmid it's transformation and any bacteria which does not have the plasmid it's non-transformative it's non-transformative okay so this one is having the plasmid you doesn't know now about the plasmid so out of this transformant out of this transformant some of the plasmid some of the plasmids will be having the gene of interest and some of the plasmid they are just like that they do not have the gene of interest so the one which have the gene of the interest which is which have the recombinant dna these are recombinants these are recombinants they have the gene of interest so any bacteria which have a gene of interest in it you call it as transformatory component and the one which doesn't have the recombinant gene but have the plasmid is transformed and non-recombinant and the one which doesn't have a plasmid is a non-transformative so now you have you know these three types of bacterias in in the that plate you are culturing so you the one which is of use is the recombinant transformation you want to identify which bacteria is transmitted and which is non-transformative so let's see what you will do you will perform an experiment so for example you have this master plate and this is the plate where you are growing the colonies you are growing the colonies of the bacteria in the agar medium in the agar medium okay so now as you know any any any of the bacteria if it does have the plasmin in it it will be having the uh antibiotic resistant genes yes yes or not so let's see here so i am using this plasmid and i have used this restriction enzyme i have used this restriction enzyme to add my gene of interest here so i have added my gene of interest here okay i have added my gene here when i am cutting the tetracyclic resistant gene i am cutting the tetracycline resistance gene so when i cut it the tetracycline resistant gene will lose its identity now it will not work what is the function of tetracycline resistant gene it will protect the bacteria from the tetracycline so when if any bacteria have pb3 pbr322 it will not be able to die or the antibiotics which antibiotics ampicillin and tetracycline will not be able to kill that bacteria if i have the bacteria with the pbr322 as these have these two genes i gave it ampicillin i gave it tetracycline the bacteria will live happily because it has these genes and these gene help the bacteria to live in the presence of these antibiotics okay now if i'm using bam h1 i'm cutting it the tetracycline will lose its identity and now if i give now if i give the tetracycline to the bacteria with this type of plasmid the bacteria will die why because a tetracycline resistant gene due to cutting has loses identity it has become non-functional and now at this point i've added my new gene of interest i've had it my new gene of interest or insert because you are inserting new gene in another gene as a result this gene is losing its identity you call it as insertional inactivation what you call it as insertional inactivation so by using insertional inactivation by using insertional inactivation you can identify which is recombinant which is non-recombinant so out of this some of them they are having the these are having the plasmid some doesn't have plasmid some have recombinant dna some doesn't have so now let's find out since i'm using bam h1 and i'm using tetracycline resistant gene uh for inserting the another gene the tetracycline resistant gene when it will get the insert or my desired gene or dna it will lose its identity it will lose its identity so when i feed when i'll feed these bacterias with ampicillin with ampicillin how many of them will die how many of them die yes how many of them will die for example i have this bacterial colonies okay so these two colonies they died these two colonies died okay now what i get from this what does i get from this so the bacterius which doesn't have a plasmid okay this is a plasmid out of this plasmid i have this ampersand resistant gene and i have this tetracycline resistant gene i'm adding my gene here i'm adding my gene here okay so any bacteria which have the plasmid whether it's the combinator non-recombinant it will survive in the present of ambisline it will survive in the presence of empicillin so here so here when i added the ampicillin these two which died they were non-transformative they doesn't have the plasmid they were non transfer meant it doesn't have the plasmid they doesn't have the plasmid okay now i added tetracycline what did i added i added tetracycline i added tetracycline now which of the above one diet for example these two diet only these two are left from this i got to know the one which died the one which died these two when you added tetracycline they died why did they die they died because their tetracycline resistant gene was inactivated due to presence of another gene that means if there is another gene they are recombinant so the one which died the one which died which one these two they died these are transformant recombinant these are transformatory component and these are the one which i need which i need for my further gene cloning okay so this is a master plate you never perform the experiment on the master plate you always take a replica i have taken another replica i have taken another replica for example this is a master plate this is a master plate i have taken the replica so this is a not the master plate this is the replicated plate okay how do you replicate just like a stamp i have a stamp i took the stamp from here to here so when i am putting or stamping here some of the bacterias colonies they will attach to the stamp and now i am getting the replication on the another one so this is how i get the replicated plate replicated plate okay so here i have got some of these colonies okay on the master plate so now i get got to know that these two colonies are the one which i actually wanted because they have the insert so these one i will grow later on for gene cloning i will grow and make my product which i want to make okay so this is how you can identify which is recombinant and non-recombinant okay since uh the one which is having the one which is having the plasmid it will be having emphysema resistant gene it will survive in the presence of ampicillin the one which doesn't have the plasmid at all it will not uh uh it will not survive in the presence of ambisline now the one which have the insert its tetracycline resistant gene has been inactivated so when i added tetracycline resistance or when i added tetracycline antibiotic to this bacteria if i'm giving tetracycline to this bacteria this bacteria will die why this will die this will die this will survive when it will survive this will survive in ampicillin and tetracycline this will die in tetracycline but it will survive the empision why because we have we have put the insert in the tetracycline if we do in another way if i am putting my insert in the impislin then what will happen then i will perform the experiment in other way i will first put the tetracycline antibiotic and then i will use the ampersand because i'm adding the insert into the empress line all right okay anyway so this is how by using pbr322 we can use it as a vector and replicate our gene and this is how because it has selectable marker as ampicillin and tetracycline resistant gene you can by using this technique insertional inactivation you can find out which are recombinant and which are non recombinant all right moving further to the next that is blue white selection blue white selection what is this so when you're using another vector what is that vector when i'm for example using another vector which is it puc18 now i have another vector p u c one eight p means plasmid uses university of california where it was uh discovered it was produced okay now this one is having one this one is having one ampicillin resistant gene this is ampicillin resistant gene and it has one lac z gene what is this lag set and laxative gene have a multiple cloning sites in it it has multiple cloning site to cut so this is always better than pbr322 why because in pbr322 you need to make a lot of replicas you want you need to add a lot of antibiotics and that's a cumbersome process it's really really untidy you can get infections as well the culture can get not you the culture can get infections as well but this is little simpler one you don't have to take replica again and again what you do is in this one in this one we use a substrate rather than using more of the antibiotics only one antibiotic is used to check the transformation in the non transformates otherwise the recombinant and non recombinant are identified based on the laxative gene the laxat gene produce the enzyme beta galactosidase beta galactosidase the beta galactosidase it converts a white substrate into blue a white substrate into blue product okay all right now what happen is now what happened is what do i have one i have transformed another are non-transmit okay now one are non-transformative another transformation in transoment we in transmit we have a bacteria with a plasmid okay and here we don't have a bacteria with plasmids so when i added ampicillin when i added ampicillin this one will survive this one will die why because it has the ampicillin resistant gene it has a plasmid now i need to identify i need to identify now which one is the recombinant which is a recombinant okay now the transformants are of two types the transformants are of two type one is recombinant another is non-recombinant okay so here this is ended here so here it is non-recombinant so now what i added i added the uh substrate so the one which have which have the insert it will be here which have the insert it will destroy the laxative gene and when the lagzi gene is destroyed there will be no beta galactosidase so when i added the product when i added the sorry when i added the substrate no product will be formed and the colonies will appear white the colonies will appear white you get it if i have a recombinant dna if i have recombinant plasmid it will be having the insert here and lag that will lose its identity it will not produce beta galactosidase so substrate will not be converted into blue color product and the colonies will appear white due to presence of substrate okay so the recombinant colonies will appear white whereas non-recombinant will not be having gene of interest lag that will not lose its identity beta galactosidase will be formed now substrate is converted into product because beta galactosidase is forming here so the non-recombinant will appear blue so any colony which is of white color i will extract it out and i will grow it to form my gene so you can use any of the vector you can use any of the technique to find out which is recombinant and which is non recombinant got it yes i think it's quite simple if you don't get it reverse rewind listen to me again because you know that's the thing for the first time it appears like oh my god what's happening but it's quite obvious and it's quite okay don't you know don't get uh anxious in it oh my god that stuff it's simple once you get it once you strike it everything is simple it all depends upon whether it has the gene in it if it has a gene that a gene will be lost okay all right so that's about uh the plasmids let's talk about another vectors we also use some bacteriophages as vectors for example m13 lambda these are viruses and they use e coli as host then we have cosmet so we have produced it artificially cosmet is between the fudge mid it's sorry it is between the plasmid and bacteriophage so we are using good characters of plasmid as well as bacteria and we are joining it and forming a new vector again its host is e coli then we have artificially made vectors back in yak you use this in human genome project and their host is saccharomyces cerevisiae that's a yeast so the host here is the prokaryotes most here are prokaryotes whereas here it is eukaryote if i am producing any eukaryotic i have eukaryotic gene what is my first preference my first preference will be a yeast because human genome project and we are eukaryotes so we will definitely use the yeast okay then we have sub vectors for plants yes we do have and this is quite important please listen to me very carefully we have bacterium agrobacterium tumefaciens agrobacterium fumification we have this bacteria agrobacterium tumifications this bacteria it infects dicot plants which plants only dicots so you can use it in dicot plants only not in monocoats this bacteria have a plasmid it has ti plasmid and ti plasmid have tdna and this dna helps in infection helps in infection what does it do it causes crown gall disease it infects dicot plant and causes crown gall disease so if i insert any gene of interest in this plasmid i can easily let that plasmid let the plasmid to enter into my plant and my plant will get that gene for example this plasmid is going to it this bacteria has the property to infect plants but while it is infecting i'm telling you are going to the plant please take this gene along with it so what i have done i have modified the plasmid i have modified the plasmid i have removed all the virulent things on it which can destroy the plant but at that place i have added i am smart i have added good genes i subtracted the bad genes in that plasmid i added good genes and now it it is the bacteria doesn't know that i have done this the bacteria will take the plasmid it will infect the plant while infecting it is not giving the bad gene it is giving the good genes so this is how you can use in modifying your plants as well then we have for us yes we do have for us something retroviruses retroviruses the disarmed one because retroviruses are very bad for example the scoville virus is retrovirus hiv is retrovirus so we have disarmed them what have we done disarmed retroviruses they are used in human to deliver a gene so we use them in animals basically in animals we have taken all the bad genes and we are adding the good genes there and then the virus will infect us it will give us the good genes okay so this is how there are various vectors we use them according to the host according to everything all right okay now let's see how it is done let's see about the processes so the first is isolation of genetic material that's dna so for example i have this cell that is a beta cell and i want to extract a particular segment of dna from it first of all what i'll do i will i will culture these cells i want a number of genes i need a number of dna so i need to have a lot of cells i'll culture them now i will treat it with enzymes right i will treat it with enzyme for example for example if this is the plant cell i will use cellulase it's normal cell i will use lipase because it's phospholipid bilayer the membrane we need to invade into so we need to disintegrate the entire okay so if it is fungus i will use chitinas so depending upon the organism i will use the enzyme to dissociate the entire cell so that its dna gets exposed but after it is done i want to destroy all the free rna there now i will add rna as well rnas as well that will destroy all the rna and some proteases that will also destroy the proteins fine now what i will see i will say i'll get a concentrate here so i want more thick concentrate i will add chil ethanol i will add chilled ethanol very cold ethanol now i will get dna in the form of threads in the form of threads now i can see i can easily see and visualize the white color threads of dna in this small while okay what is a while that glass container like this i will see when i added ethanol the threads are formed there okay so what are you technically doing here by adding the chill ethanol you are precipitating the dna as i've told you concentrating or precipitating okay so here what we have done we have precipitated the dna so now the dna can come in the form of threads all right moving further to the next now i want the few segments of gene for example i got to know that particular gene is near to the restriction site of eco r1 so i will use a restriction enzyme to cut my dna into various fragments okay so i have the huge dna i will add restriction enzyme i will add restriction enzyme and that restriction enzyme it will cut the dna but now i have got a lot of fragments i have got a lot of fragments now i want to see which of these fragment is the one i desire or the one i want so for that i need to separate that i first of all need to separate that and then isolate so now the next step is separation and isolation i want to separate and isolate separation and isolation or specific dna okay you got my point so here we have a huge dna huge dna i added restriction enzyme it has chopped it at some places like this now i have got small small pieces of dna but which is the one i desired which is the one i desired out of all these i want to get it out so for separation for separation i will use a technique that is gel electrophoresis gel because i am using a gel here which gel agarose gel i am using agarose gel from seaweed from red algae this aggro gel is extracted from seaweed the algae okay electrophoresis because i'm using electric field here electric field here okay now what i do i have this plate and this plate is full of agarose gel this plate is full of agarose gel okay so here i have attached anode what is it it's anode and anode is positively charged anode is positively charged your dna has negative charge dna is negatively charged okay so on the basis of size we are separating the dna and we using the property of dna that it is negatively charged we want to separate the dna because we want to see which one is the one i need and i know its molecular weight i know the molecular weight of the dna on the basis of molecular weight i will separate all the strand then i will pick up which is my desirable of that particular weight okay so i'm separating the dna based upon its molecular weight how because this agros gel it has pores what is it has it has pores so when i put my dna sample here i have put my dna sample here this is dna okay and dna is negatively charged now i plugged this uh anode in when i plugged it in the dna based upon its molecular weight it will travel downward it will travel downward this size will of the smallest dna and this is the largest dna here i will get small bands of dna like this okay and here we have a ladder that ladder tells about the molecular weight just like a scale like you have a scale to measure your height just like that we have leader for example this one is of like 2 kb so i have isolated it from the gel i have isolated it from the gel okay so this is all with the help of this technique which technique gel electrophoresis we have separated the dna based upon its molecular weight so which is the smallest one it will move the more away from this position okay so that's why it's written in ncrt smaller the fragment farther it move smaller the fragment farther it will move further it moves okay the away it will move and how are we separating it we are separating on the basis of sieving effect which effect sieving what is the save that you use at home to filter the tea you have that sieve now let's see when you strain the uh for straining the tea you use a sieve okay that see when you strain the tea the large molecular size tea particles they remain in that sieve and the tea flows off in your cup just like that these small you know small particles of dna which are of larger size they will get stuck in the pores of agarose gel so because here agarose gel spores are acting as a sieve right so this is how they are moving downward for example uh if we have these pores like this okay the smaller one the smaller one can easily get through it but the bigger one will get stuck here so this is how you call it as a sieving effect okay so by this you have separated the dna but now how you are going to isolate it for isolation we have a technique known as illusion illusion means when you cut the fragment from gel you cut the fragment from gel and you take it out you call it as illusion so after you know this is my desired dna you will cut this gel piece from here and you will take it out and that is known as illusion so here what we have done we have isolated the specific dna we have we have done it with the help of illusion okay but when you are isolating the dna strand that you are doing it with the help of spooling so here we have added the chilled ethanol so that the dna stands will be formed and you can easily isolate it this is known as spooling this is known as spooling whereas when you are taking the specific dna here it was just a genetic material dna but here is a specific dna you are taking out and that process is illusion both are isolation but that's of the entire genetic material but this one is of a specific dna okay so now as if we have you know uh done this separation right so now i've told you we we can also isolate it but how can you see it here i have drawn of the blue color that's so you can see the bands but what if i draw the bands in the black color can you see it this is how the dna looks like the agarose gel is of white transparent color and the dna there also looks like one white transparent so you can't see the dna with naked eye so for visualizing the bands what do we do we dip this gel in uh in a dye and what's that dye that's ethereum bromide so we have uh this uh type of a gel here you cannot visualize the dna because it's also of the same color now i will dip it in the ethereum bromide ethereum bromide is a dye okay now what i will do after i have dipped it i will pass the uv rays i will pass the uv rays so when i pass the uv rays i can see orange dark orange dark orange color bands and this is how the dna looks like so you cannot see or cannot visualize the dna with naked eye for that you need to have this dye so this dye after you pass uv rays only will you be able to see the bands you can visualize the band and which color dark orange color dark orange color dna band all right okay guys moving further to next so for multiplication we have another technique and that's pcr what is pcr polymerase chain reaction polymerase chain reaction this is a very simple technique this is a very simple technique here we have a thermal cycler a small machine and in that machine we add all our sample how what does it sample contain it contain dna template that you want to multiply you want to replicate we add dna polymerase there because we need enzyme which will able to multiply it we will add our primers what are these primers these are oligonucleotides that means nucleotides the chain of nucleotide around 5 to 8 nucleotide that will act as primers which will initiate the replication process and then certain enzymes or irons not enzymes certain ions okay so the first step of the pcr is denaturation what is the first step here here you can see we are heating it okay so the first step is denaturation denaturation takes place with the hell at the temperature of 94 degrees celsius when you give high temperature to a dna strand its hydrogen bonds will break and both the strands will separate both the sands will separate now it will lead to the another it will lead to another step that is annealing in any link the primers you can see here the small nucleotide they are going to added specific location you are adding random primers you don't know about it right so we are adding random primers wherever they will find the complementary part they will attach there they're around five to three prime and the three prime is always you know free now now we have added dna polymerase now which dna polymerase stack polymerase tag polymerase tag polymerase is a type of dna polymerase that has been that has been isolated from thermos aquaticus thermos aquaticus is a bacterium it is a bacterium uh you know which is you found them in in the thermal springs hot springs because they can survive at such a high temperature that's why we have used it because here you are going 94 degrees celsius if you add because we are adding all the material at once if you add our dna polymerase it will denature the enzyme will destroy and it will not be able to multiply your dna okay so the next step is annealing because their primers are adding now the enzyme will work and it will replicate and you know your dna will replicate okay so here that comes the next step that is extension next step is extension so there are three steps in the the pcr reactions first is denaturation where you are separating the strand then is annealing you are adding the primers and then extension where you are replicating the dna this takes place at 50 to 60 degrees celsius whereas extension takes place at around 72 degree celsius and then around after 30 cycles you can get around 1 billion time of the original dna see this is how you can amplify it amplify means you can multiply the desired specific dna which you want to add into your host okay all right so that's now you have multiplied the gene what's next now you want that gene to get into your host so for that we have various technique so the first technique is a very simple technique that's transformation that's transformation okay so first of all we are assuming we have we have isolated the gene we have amplified it we have also joined it with the vector we are already assuming we have jointed with the vector and that vector is plasmid so what we do is in the transformation technique we have this bacteria first of all we will treat the bacteria with calcium chloride this is how the bacteria will become competent and it can easily bind with the plasmid here this is plasmid this is plasmid this is competent host competent means this is now ready to take the plasmid okay competent means it is ready to take the plasmid now we will give it heat shock we will give it heat shock of around 42 degrees celsius and due to heat shock the membrane permeability of bacteria will change membrane permeability of bacteria will change its pore will open its pore will open and plasmid will enter and again you keep it in a chill environment okay so this is how you can let the vectors enter into your host this is one technique other technique is micro injection you can directly inject you can directly inject the gene of interest into your host for example this is your host you can directly inject to the help of micro injection the desired gene you want and this is usually done in animals the one technique you already know that is performed in ivf icsi intracytoplasmic sperm injection this is also like that only you are giving the particular nucleus to the ovum so this can also be done during uh you know when you want the desired gene product another technique which you perform in plants is biologics biolistics what is biolystics gene gun so where do you perform it you perform it in plants so we have a gun we have a gun for example this is a gun okay and in this gun you coat the dna with the inert substance like gold or tungsten you have this gold or tungsten and then you have dna in it okay so we am writing dna coated with gold or tungsten okay now just like any gun it also has a trigger this is very bad gun i have made so it's not like that you can google it if you want to so when we press the trigger this is now fired up and it directly enters into the cell like this okay so this is a gun technique where you just push the trigger and the thing gets shooted to the cell directly otherwise you also use a technique electroporation what we done in electroporation so the name is telling everything you give electric field to the host and its pores get open like in the previous we have given the heat shock and the pores get open in the electroporation electric field is given to open pores and now easily easily the vector can enter inside the host so any of the technique depending upon vector and host we can use it depending upon your availability of product we can use any of the techniques so that the recombinant which has been made the vector that has been uh been ready it can easily enter into the host why there is a need to enter into the host because if it doesn't enter into the host it will not be able to multiply it will not be able to multiply let's see what's next so now we want the gene product it has entered into the host okay so the the desired dna has been entered into the host first of all we will identify we will identify recombinants one by the techniques we have discussed the blue white selection or all we will identify the recombinant one okay we will culture them we will culture them in large scale we will culture them in large scale when we culture them in large scale we need something which are known as bioreactors what are bioreactors while traveling around these cities you know at the countryside you must have seen large factories with large steel vessels right in the beauties you must have seen that or not if not it's fine so bioreactors are large vessels what are these large vessels how much large they can they have a capacity of 100 to 1000 liters they can store this much of a culture brought in them okay so we will produce the products at a large scale we will produce the products at a large scale by using bioreactors now these bioreactors they were earlier known as fermenters how they are different so there are two things one are bioreactors another are fermenters for mentors are technically some vessels where anaerobic conditions are created but when you use bioreactors when you use bioreactor both anaerobic and aerobic culture can be present in short form i can say the fermenters are a type of bioreactors the fermenters are a type of bioreactor then why the fermenter term is used because earlier the biotechnology this was all related with fermentation alcohol fermentation right curd fermentation everything like that so nowadays we have use a wider term because we are not restricted to anaerobic things we are just we are also doing something where aerobic organisms are giving us a lot of products okay so that's why these large large vessels are known as bio reactors so the culture you which you are doing in a large scale it can be of two types okay what is culture the growing of bacterias this culture can be of two type one is the open type and another is a closed type okay the close type is discontinuous and this one is a continuous culture how they are different in this i have a bioreactor i put a fixed concentration of my culture and then i close the lid i close the lid okay i let it i let it close for some duration i know after 15 day the product will form i will not touch it for around 15 day after 15 day i will open it take out my products and then i will wash the vessel for the next batch so that's why it is also known as batch culture what do you call it as batch culture okay so here we are putting the product in different batches so one batch of 15 days next batch for 15 days so what is done here we have fixed quantity we have fixed quantity of culture we have fixed quantity of culture here and now we will not touching it and it is done in batches it is done in batches what's the side effect because you're not touching it the product is formed here only because it contains a lot of bacterias and bacterias will be producing waste and when the bacterias produce waste and when you don't remove the waste what happen is their growth will reduce their growth will reduce okay but here in the continuous what we are doing you have this uh bioreactor at one side you are adding you are adding the culture on the another side you are removing the product okay you're adding the culture you're removing the product when you're removing the product the waste will be removed waste removed waste will be removed as a result all the bacterias which are here all the bacterias are there they are in a bacteria are in exponential phase that means because they have nothing to inhibit them they have nothing to inhibit them they will always be in a log phase or expo financial phase all right so this is the difference between these two in open one you're continuously adding culture you're removing the product there is no fixed concentration of culture it's coming going coming going and bacterias will not be having a lot of waste here because it's a flow when something flows when you have a stagnant water it's always dirty but you have a river with flowing water it's always clean because it's flowing it's not stagnant just like here it's a river from one side culture is added from another product has been taken out but in this we have a fixed culture and here the bacterias are in a lag phase and here there will be a lot of waste product as well okay all right now moving further to next how the bioreactors they look like so there are two type of bioreactor you can see in your ncrt the first is simple start another is path start how they're different here you can see the bioreactor have a curved base this one is having curve base it is having a curved base and this curve base allow a better mixing if anything is flat it will not be able to mix properly but if it has a curve fix it can easily move also you can see there is a steam for sterilization because you want nothing else get uh you know grow there because bacterias are there they have a food fungus can easily come right so you don't want fungus to grow here so we we are also putting the sterilization process here also we have a motor to move this blades to move these blades so that it can easily you know these are like stirrers they can easily mix the products then we have acid and base control because bacterias have you know certain things they can only grow in acidic condition they can only go in basic condition so here we have something to control the ph as well then we have form breakers because you know when if you have seen beer the drinking beer yes alcohol so it has form in it so if i'm producing something if the product formed is just little one and everything is the form there will it is uh justified for me to produce uh that no i don't want form i want product so here sometimes during the culture period of the bacterias and all a lot of form is formed so we want to break the form and we want product so there is a form breaker which will uh break the excessive form okay all right then we have another type which is part stir tank how it is different it has this thing which is a sparger what is it it's a sparger if i um if i show you how the spaja looks like here it is like a plate with hole so when you move the sparger this is like how sparger is when you move the sparger like this it will form the small bubbles and these small bubbles are there and they will increase the surface area for oxygen transfer the big bubbles they form obstruction for oxygen whereas small bubbles will bring more oxygen so that's where sponge is very much important when you're performing aerobic cultures for the bacterias which perform aerobic respiration all right so this is how the bioreactors they look like so do do do remember how do they look like okay then now we have uh form the product okay we have formed the product so next is the downstream processing so whatsoever we have done before they were all in upstream now comes a downstream in downstream what we do we will isolate the product first we will isolate the product after isolation of the product we will store the product we will pack the product we will store them at appropriate temperature for example you produce a medicine and medicines need to be refrigerated so you will produce you will store according to them and after storing it you will market it you will store it you will pack it okay you will market it and you will sell it according to your needs so this is how a product is formed and you have earned the money as well today so you're rich now okay anyways um so this was how we produce the products there let's solve question let's all question i know you have become rich today you have produced insulin and you have marketed as well but uh let's come back to the ground reality so first the correct order of steps in polymerase chain reaction do it very simple very simple tell me these are these all are pyqs these all are pyqu's previous year questions quite simple yes okay so the first was denaturation you were destroying you were destroying the two strands okay not destroying you were separating the two strands that's denaturation at 94 degrees celsius and then you add the primer that's annealing and then dna polymerase will work the tag polymerase and that's extension so the answer is c okay next a gene whose expression helps to identify transform cell is known as so you need to see whether the cell is transformed or not for that you what what we used we use empisin resistant genes so these were a type of what they were a type of selectable markers so answer is a next dna fragments are positively charged negatively neutral either positively or negatively charged depending on the size no dna is always always negatively charged so that's why you can perform gel electrophoresis in a better way if it hasn't been like a differently charged it will be difficult okay so answer is b next the dna fragments separated on agrocell can be visualized after staining with very good question okay so here here when you have uh okay where does it go okay that's the question so here when performing gel electrophoresis i told you i told you that the dna you can you cannot visualize it under the gel you can only visualize when you put a stain on it and that stain works when you pass that gel through the uv light okay so what was that ethereum bromide so the answer is all right next question the two antibiotic resistant genes on vector pbr322 very simple because i know you know the structure of pbr32 now that's ampicillin and tetracycline so answer is p though these all are type of the antibiotic resistant genes as a selectable marker but particularly on pbr 322 ampicillin and tetracycline next which one of the following equipments is essential uh required for growing microbes on a large scale for industrial production of enzyme as i've told you when you want to culture in at the large scale in the factory or something like that you always what do you need you know large vessels and these large vessels they are around 100 to 1000 liter big volume so that's bioreactors the d4 diksham all right next dna precipitation out of a mixture of biomolecules can be achieved by treatment with you want to concentrate you want to precipitate the dna so what was there for that we need chilled ethanol so answer is b all right so that's it about the biotechnology principle processes i will meet you in the next class till then bye bye study hard it's quite you know different type of a chapter so sometime your brain doesn't adapt to it so don't worry because everything was theoretical it was like human and all but this is a little different experimental chapter so it's very interesting just go for it read it again and again you will get it for sure bye bye thank you take care