[Music] foreign [Music] [Music] foreign [Music] and what we were discussing we were discussing about the different properties of the enzyme in the course enzyme science and technology so for what we have discussed we have discussed about the development of the history of the enzyme history of the enzymology then subsequent to that in the module 2 we have discussed about the structure of the enzyme so uh while we were discussing about the structure of the enzyme we discussed about the primary structure secondary structure tertiary structure and quaternary structures and we have discussed several techniques which you can actually be able to use for uh you know determining the uh these structures of the enzyme in the previous module we have discussed about how you can be able to isolate a gene from the uh from the from the genome whether you are aware of the genome genomic sequences or whether you are unaware of the genomic sequences you can be able to use multiple approaches to isolate the gene of your interest which is going to code for the enzyme of your interest and then once you have got the fragment we have also discussed in the previous lecture how you can be able to clone that fragment into the vector of your choice now once you have cloned the fragment of your choice into the into the vector the vector has to be delivered into the suitable host so in the today's lecture we are going to discuss about the various strategies what you can actually be able to use for delivering the DNA into the suitable host so uh what you can see is this is the scheme for enzyme production right and in this scheme we have already discussed many aspects of this scheme where we have said that the you are going to isolate the gene fragment from genome right either you are aware of the genomic sequences or you are unaware of the genomic sequences this means either you will use the genomic Library approach or the cdna approach or you are going to use the PCR once you have these genomic sequences then you are going to perform the decision digestion of this particular sequence and that's how you are going to get the sticky ends same you are going to do for the vectors right in this case we have taken an example of the plasmid which is the bacterial vector and then you are going to put the rotation digestion and you are going to have the SDK ends of the plasmid then you are going to put them for the ligation reaction and that's how you are going to get the recombinant Clone this recombinant cologne is has to be delivered into the suitable also that you can be able to use that for protein production so uh when we talk about the DNA delivery in host we have to understand the many aspects of this particular process so DNA delivery in holes so you can imagine that how the people have discovered or how the people have get to know about the DNA delivery host so you can imagine that you have a donor cell from which you are actually going to have the uh you know the DNA molecules and these DNA molecules are you know that the negatively charged so these DNA the charge on the DNA is negative charge on the DNA molecule interacts with the whole cell especially with the cell surface which means if I change the cell surface chemistry of this donor cell in such a way that it is actually going to take up this DNA and they will go and you know stick to this particular you know cell wall or the plasma membrane they will be going to taken up by the cell so the surface chemistry of the whole cell so surf host cell surface chemistry either will attract or the Ripple DNA as a result of the opposite or the similar charges which means the cell surface of the host is either going to so be positively charged or the negatively charged if it is positively charged it is actually going to attract the DNA if it is negatively charged it is going to repel the DNA so presence of the cell wall in case of bacteria or fungus or the plant causes the additional physical barrier to the cellular uptake and the cellular entry so actually the entry of the DNA is not a facilitated process it has it has multiple barriers and multiple hurdles and all these hurdle has to be overcome by preparing our computers so what is mean by the competent cell is the cell which is actually going to be readily be taken up the uh so which is readily going to take up the okay and you are supposed to prepare the computers which means you are not a wild type cells are not competent enough they are not going to take up the DNA but you can actually be able to uh you know treat them in such a way that it's going to change the surface chemistry of the cell surface and that's how it is actually going to take up the cells uh naturally uh the this process is called as the transformation which means the process in which the DNA fragment from the one cell is going to be taken up by the under cell so you can imagine that if the donor cell is resistance for the antibiotic for example you have taken an antibiotic and that has been you know some some bacterial cells are actually acquired the resistance for that but antibiotic cells by many mean because of some presence of some DNA right so if you have a presence of DNA which is responsible for providing the antibiotic races this particular regular donor cell is actually going to be overcome or it is actually going to be overcome from the antibiotic mediated killing so in that case what it will do is it will actually going to throw this particular DNA and this DNA is going to be very very valuable for the other bacterial uh members right so what they will do is they will going to accept by the other cells right and as soon as the other cell is going to take up this DNA it is actually going to also acquire the system and this is actually the problem way in which the transformation is actually going to be uh you know going to help the single bacteria to acquire the resistance and also to spread the resistance throughout the colony how it happens it happens that you are actually going to have the donor cell from the donor cell these the donor DNA or the DNA fragment which is actually going to have the antibiotic resistance genes or any kind of phenotypic gene is actually going to put into the extracellular media and then this extracellular media is actually going to interact with the acceptor cells so you are going to have the competent recipient cells so that donor DNA fragment will bind to the competent recipient cells and that's how it is actually going to be taken up so mechanism of transformation is a process by which the cell free DNA is taken up by the another bacteria the DNA from the donor bacteria binds to the competent recipient cells and DNA enters into the cell the DNA enters into the recipient cell through a uncharacterized mechanism the DNA is integrated into the chromosomal DNA through a homolographic combinations naturally transformation is common between the closely related species which means there are uh you know so natural transformation we have already taken an example where the you can actually be able to use that for transferring the resistance from the resistance or the phenotype from the pencils one bacterial population to another bacterial population but the in the lab what you are going to do is you are going to add the excess chromosomal DNA or you are going to add the recombinant Clone uh recombinant DNA and in presence and when you add this recombinant DNA to the competent the CPN cells the DNA is actually going to interact with the computer recipient cells and that's how it is actually going to taken up by the cell and once it is going to taken up by the cell it is actually going to go for cell division and that's how you're going to have the transform cells so where the copy is on the both side of the DNA sometimes you actually going to have the integration sites on these DNA so they will integrate into the genome otherwise they will remain as the extra chromosomal DNA in the form of the plasmids now how you are going to prepare the competent cells for your laboratory experiments depending on the bacterial species you can be able to use the different types of chemical reagents which are actually going to change the surface chemistry and will make the cell competent so that they will take up the new cells they will take up the uh that XFL DNA so for example in the case of statofocus new Mini you are going to treat the cells with the mitomycin c or fluoroquine Norm so when you treat the cells with mitomycin C or fluorophenol they are going to be competent and they will actually going to take up the extra cellular DNA similarly you have the bacillus subtitles you are going to treat the cells with the UV light and that actually is going to induce or that is going to change the surface chemistry and that's how it is actually going to be computer solves in the case of helicobacter pylori the bacteria which is responsible for the ulcer right or it's going to be treated with the ciprofloxacin ciprofloxacin is an antibiotic okay so the treatments are different because your surface chemistry is going to be different the physiology of that particular bacteria is going to be different so that's why you are actually going to treat it with the different competent agents then we have the living then that is actually going to be make the competent by treating you with the mitomycin c norfloxacin ofloxacin and all these kind of antibiotics and hydroxy urea as well as uvulite for example E coli which is the called as laboratory stain so this is the lab strain and that can be competed by treating with the covalent chemicals like the calcium chloride and rubidium chloride and the cells are how you're going to make the competent cells so you're going to treat the cells with the different agents so all of these agents so just for sake of how we can be able to make the competent salt I am giving you an example of the E coli cells so we in in the E coli cells we are going to take an example of how you can be able to use the calcium chloride to prepare the E coli computer cells so preparation of the competent cells so bacteria is incubated with the divalent cation like the calcium chloride magnesium chloride or the rubidium chloride for 30 minutes at 4 degree celsius so first you are going to grow the bacteria and then your bacteria is going to incubate it with a divalent cation such as calcium chloride magnetic chloride or rubidium chloride for 30 minutes at 4 degree celsius what will happen in this period during this process the cell wall of the treated bacteria is going to swell and it gather the factor required for the intake of the DNA docked onto the plasma membrane uh so you can imagine that when you are doing all this preparation it is actually going to take up the calcium chloride or the divalent cations and these divalent cation table what they are going to do is they are actually going to take where they are going to make the cells a little fragile okay so in that case you might have to take care of these cells very nicely because there also get susceptible for any kind of shear stress for shear stress is a is means that you are actually going to suppose uh when you are swimming into the air water right the water whatever the stress you will feel when the water is actually hitting you that is called as a shear stress shear stress means you have two layers and these two layers are actually rubbing to each other and that is actually being responsible for causing a friction and that is actually a called as shear stress so they are very susceptible for shear stress for example if you spend them a very high speed uh you probably will actually going to like the results so that's why these competent cells are very fragile you cannot run that very high speeds okay and you also cannot do the pipetting at a very high pipetting right for example if you do a pipetting a lot of you know with the thin bore tips it is actually going to destroy the cells because it's going to life the cells uh how you're going to store the computer cells you can once your computer cell preparation is over then you can actually be able to add the 15 to 20 percent glycerol and you can store it at minus 80 degrees Celsius and whenever you require so 15 to 20 glycerol is actually going to work as anti-freezing agent right so it's not going to allow the formation of the water crystal and that's why it is actually going to protect the bacteria from getting the any kind of damages ah so you have to be very careful that the cells are very fragile they are sensitive for the centrifugations and they are also sensitive for the pipe eating that's why you can once you prepare the computer cells you allocate them in a you know in into a suitable volume and then you can just use the same that volume completely you cannot just pipette it out and prepare the more early chords ah how are you going to prepare the competent cells so what you can do is first you are going to do grow the bacteria okay so if you see the bacterial growth curve what you're going to see is it has the lag phase it is going to have log face it has the stationary phase and it has the death phase or the decline phase so the cells which are in the lock phase stationary phase or the cells which are in the decline phase are actually under the steam stress which means these three phase of the cell right whether you are under the log phase or the stationary phase or the depth phase are not suitable for preparing the computer cells so you have to grow the bacteria in such a way that it should be in a lock phase you know in a logarithmic phase so that they are very healthy they are actually acquiring the different types of factors and there is a no dirt of nutrition so there is no ah loss of nutrition so they are actually going to have lot of nutrition and they are actually having the ability to produce lot of factors so that is actually going to be the best bacterial culture what you're going to use so the growth stage of the bacteria has a significant impact for its ability to take up the foreign DNA the bacteria at lock phase is more active and efficient to perform the DNA damage and repair than the stationary phase as a result it is preferred to use a bacteria of log face for making the competent cells for the transformation so once you grow the bacteria bring it to the lock phase and then you ah you know collect the law of phase bacteria and then you are going to use that for the competent for Preparation so how you are going to do the transformation so on the day of transformation competent cells are incubated with the DNA or the circular plasmid containing appropriate resistance genes such as MPC in resistant genes for the 30 minutes on ice so first here what you are going to do is this is the step one number one so in the step one what you are going to do is you are going to throw the competences right because remember that the computer cells are going to be stored at minus 80 degree Celsius in the 15 to 20 percent glycerol right so first what you are going to do is you are going to thaw the cells and you are going to use that for transformations then in the step one you are going to incubate the uh the DNA either the linear DNA or the circular plasmid with in the eyes right along with the for 30 minutes so and then we are going to add the basically in the resistance okay and then once this step is over so you are going to have the 30 minutes incubation and once the 30 minutes incubation is over then you are going to give the heat shock so in the step two you are going to do a heat show so competent cells are given a brief heat shock such as 42 degree Celsius for 90 seconds to relax the cell wall and high temperatures stress causes upregulation of the factor responsible for DNA recombination and repair so then what you're going to do is you're going to give the heat shock so in the heat shock step what will happen is that because you are giving the heat shock the cell is actually going to swell and it is actually going to take up the DNA into that and because you are providing the heat shock it also going to induce the production of the cellular Machinery which is a source bill for DNA recombination and repair because that is very very important event because if the even if the it has taken up the DNA and it cannot actually be able to recover from the damages that the cell is actually going to die then in the next step what you are going to do is you are going to add once the heat shock is over right then you are going to add a chilled media and that is actually going to bring the rigidity of the cell wall or the plasma membrane and that's how it is actually going to seal the pores what is going to be prepared into the plasma membrane and that's how it is actually going to help in terms of the faster recovery of the transform cells so once you add the chilled media you can actually allow them to recover for 30 to 45 minutes at 37 degree Celsius and after this recovery you are actually going to ah plate the cells onto the agarose plates right so after this it is plated onto the solid media with the appropriate antibiotics such as ampicillin and allowed to grow for another 18 to 24 degrees Celsius at 37 degree celsius incubator so when you are done with the you know chilled media you added the chilled media you let them to you know remain into the 37 for some time and then you are actually going to Plate them onto the lb empathy in plate so for example in this case we have taken the empathy in this Gene so when you do the ampicillin the plating architecture and then you incubate into the incubator for ah 18 to 24 hours then it is actually going to form the colonies and now these colonies are the transform colonies which have taken up the DNA okay then untransformed colonies will actually not going to have the empathy in the distance and that's what they will actually going to die in this lb amps link plate so transform cells with appropriate resistance will grow and it will give you the colonies so once you are done with the Transformations how the how the bacterial plates will look like so this is actually the untransformed or the control plate okay and this is your transform plates so what you see here is I have we have transformed a plasmid into the lb empressleen and if you count the number of colonies it is 80 to 90 colonies what we got into this particular plate whereas there is no colony in the control plate which means the plate where we have not done any ah plating which only contains the the cells so this is without DNA and this actually has plus DNA okay now using this in use this number right how many colonies you've got you can be able to calculate the transformation efficiency what is mean by transformation efficiency the number of colony forming unit obtained by transforming one microgram of DNA into a given volume of competent cells okay so the number of colonies what you got from the one microgram of DNA is called as the transformation efficiency and it is a very very important parameter to judge how good your competent cells are because if they are very bad ah you cannot use them for the cloning reactions ah for example if you transformed the one microliter of 0.09 microgram per microliter plasmid into 100 microliter of competent cells you added 900 microliter of LV to your cell to get a total reaction volume of 1000 microliter and then plated 100 microliter of the transformation the plate has 450 Colony on its next day which means ah you have actually added the this much this much amount of DNA and this much amount was the reaction volume so what you are going to do is you're first going to calculate the amount of DNA what is you have used so if you calculate this your what you are going to know is that you have plated 0.001 nanogram of DNA now if you want to calculate the efficiency 450 colonies from point zero zero five nanograms into thousand because this is what the dilution you have done and that will actually going to tell you that you have a transformation efficiency that is 4.5 into 10 Power 8 ah so that is very good actually okay so 10 to Power 8 is a very good efficiency ah sometime you may get even more 10 to power eleven and so on so that will be going to be very very high border actually so any number which is above to 10 to power 4 or 5 is reasonably ok apart from the transformation by the chemical method you can also use you can also do the elect operation so electroporation is a method where you are going to use the electrical pulses into the cell so what will happen is that when you put the electrical pulses you are actually going to make the hole into the plasma membrane and utilize the and since the DNA is already out it is actually going to enter into the cell and that's all and after that you are going to put the chilled media and it is actually going to resell this uh pore and that's how it is actually going to be taken up the DNA so the plasma membrane is composed of the lipid and protein these macromolecule gives a partial conductance to the cell membrane so when a high electrical pulse is given to the cell the charge run across the membrane and partially disturb the arrangement of the lipid molecule as a result it makes the formation of pore and allow easy passage of the macromolecule especially the charge molecule like DNA into the cell after the electroporation the cell is allowed to recover from the damage and it forms a colony on the cell active solid media so you can see that this is a plasma membrane so plasma membrane is made up of of the lipids right so lipids are arranged like this right and lipids are partially been charged so when the past plasma when there is a very high pulse which goes on top of this these lipid molecules are getting rearranged and because of that it actually allows the passage of the DNA into the cell and this DNA is so this is actually very momentarily so once you add the media this pulse is going to be over and therefore it is actually going to seal this particular thing and that's how the DNA is going to be delivered into the host and then later on you can actually allow the cells to recover and that's why you are going to have the you're going to have the transformation done if the advantage of electroporation is that it does not depends on the surface chemistry of the or cells okay it depends on the so so that's why it is very very robust compared to the chemical Transformations the disadvantage is that the electroporation is required a specialized instrument like the electroporator and it also requires the electro quits so what are affecting factors affecting the transformation efficiency the plasmid size then the form of DNA so you can you you know that when we were discussing about the plasmids uh there are three different forms either the triple free forms OC forms and the super quiet form okay so the transformation efficiency depends on the surface area of the molecule so surface area of the molecules the super coil is smaller right or smallest whereas the other two forms are actually having the larger surface area so that's why if you use the super coil DNA the transformation is she is actually going to be very high compared to the closed circular DNA or the open circular DNA similarly the plasmid size if you are working till 10 KB the transformation efficiency is going to be very high but if you go beyond that right if you go above to the 10 KB fragments the transformation as you can see is going to be very very low and in those cases you might have to use the electroporation or the other methods you cannot rely on the chemical methods then the genotype of the cell cloning strains for example E coli K12 strain have four to five times the transformation efficiency of the similar stain for linear DNA which is poorly transformed in E coli direct B correct d mutation can significantly improve the efficiency of its transformation then the third is growth of the cell so log phase cells are best for preparing the competence also then the method of transformation so we already have discussed that the chemical methods are actually going to give you the less transformation efficiency compared to the elect operations and then we have to also have the damage so expose the DNA to UV radiation is actually going to be another factor which is all affect the transformation efficiency so uh we have we have prepared a small demo clip only to show you the different steps of the Transformations and there the student have discussed the many aspects of the you know practical considerations which you can use or which you can consider while you are performing the transformations so let me take you to my lab and they will actually going to show you a demo where they are going to show you how to transform the computer cells research scholar at Department of biosciences and bioengineering and IIT development in this video we will show you how to prepare competent cells and how to transform plasma Behavior in addition to that while preparing comp cells I will show you what are the purpose we needed and how to prepare these Comforts step by step and also how to transform plasma DNA using electroporation method so let's learn how to do hello everyone in this video we will show you how to prepare competent cells and transform the blast bit and plate pieces so let's start the procedure before preparing the councils we need some inoculum this is the DH fire phynoculum I am going to inoculate in a new while for comsteps preparation so I will show you how to inoculate so now I am going to in athletic culture then we will wait [Music] that is the dark place of bacteria where you can prepare the complex that is the good stage [Music] thank you [Music] thank you after inoculation now we have to keep it in individual till we get required body so it should be 37 degrees Celsius and 150 RPM [Music] [Music] now we can see some growth in here now we have to check what is the body of this culture we can measure it in spectrophotometer foreign [Music] [Music] [Music] [Music] what is the voltage before doing that and how to plank against the so this is [Music] our this [Music] then ask but that 40 is 0.466 it is reception for our Concepts definition so we will use this culture but come since preparation then transfer so with that sufficient body next we have to send reviews the culture and palate our business after that we will put their councils using client number capture so I am going to transfer into [Music] the certificate [Music] [Music] now I am going to centrifice the culture at 4000 RPM for 10 minutes so in order to get so smart selfie thank you [Music] [Music] thank you [Music] [Music] [Music] after mixing the contents and self-related Define one molar capsule chloride we have to keep the same suspension for half an hour then you have subsequent steps with how to centrifuge and wash 50.1 molar calcium chloride for another two times after final step we have to add all right Improvement [Music] s used for storage [Music] we have watched in final step so we have to mix this [Music] [Music] [Music] foreign [Music] foreign [Music] [Music] foreign procedure which contains checking the OD like what is the observance of the semicultural for 0.42.5 uh absorbance at 600 nanometer is preferred different because at that point the sensor in exfoliation phase Exposition based sets are good for transfer transformation and we have to pellet down at 4000 RPM for five minutes the resulting pendant with resuspending 0.1 molar calcium chloride so we have to keep a nice for 30 minutes again we have to centrifuge remove the resulting calcium chloride solution and add fresh calcium chloride solution again fireman again keep for 20 minutes and centrifuge again whatever the resulting fillet you got we have to resuspending point one molar calcium chloride and allocated to 1.5 MLS so they can be stored at minus 80 degree Celsius for up to six months how to add a plasmid in aseptic condition so we kept this Concepts and attachment DNA inside [Music] we kept that now we will give a heat check at 42 degree Celsius we gave heat shop to be counted cells at 42 degree celsius and we have to keep 10 minutes on Ice then we will add Erb media cold elbi medium and foreign you need transform cells will keep an incubator shaker at 37 degree Celsius with a rotational speed of 150 ampere we just transformed a plastic DNA into capsules but during this procedure we have to be Cassius because any mechanical disturbance to the comp cells will lead to decreasing transformation efficiency so while handling the comp cells we are not going to keep our side like normal temperature always will keep cells are mice so otherwise the transformation efficiency will go down and another thing is that we should not condition and also during transformation [Music] now we got sufficient growth for transformation we will centrifice the cells and blatant suitable [Music] thank you after certification we will remove all the media but we will keep 100 micro liter resource 20 pellet and take 20 microliter and play time advantage but if you are expecting low transformation efficiency you can use all 100 microliter for rating this is the best way to increase chassis of transformation [Music] now we will take out the media and keep Hunter making this okay [Music] okay [Music] now we have plated the cells we will keep in the incubator for overnight then we will observe what is the how many calories [Music] now we can see there is a cornice appeared on the transformed plate like in but there is no colonies on only comp cells plated one so you can see colony is here now we will calculate the transformation efficiency so there is a formula for transformation efficiency number of colonies you got in that bed and that my program of DNA you used multiplied by final value matric worry how much value you recovered that is 100 microliter for the based on your chest and how much volume plated so suppose you got 500 Carnation that played you used 0.001 microgram of DNA into 100 microliter is total recorded but we plated only 20 micron means you have 2500 divided by 0.00 so that will give you 2.5 into 10 power 6 transformance per microgram of here in this video we showed you how to transform a plastic Behavior first of all we've taken by 4 OD that is log base bacteria for preparing Concepts then we prepared this compounds in 1.1 molar calcium chloride solution and the washed those competencies we used for being transformation and also for electroporation method we discussed what are the things need to be done right what are the pulse how much pulse we have to give and what time so all these things discuss very nicely hello in previous video we shown that how to transform uh plasmic DNA using calcium chloride method in this one we will show how to use an electroporator for transforming DNA so this is the cuet for uh we use for electroporation so there are two electrodes placed and this is the sharp part so we will connect here like this and all the adjustments pulse how much time we have to give the pulse all these things we can set here once the setting is over we can keep cells along with our plasmid DNA inside and then fold and we will press once so whatever the pulse generated we have given the pulse that is generated inside and through the electrodes through the electrodes it will pass us through for a minute fraction of time because of these electric pulse the pores of there are small ports found in bacteria if any plasmid adjacent to that cell happens then it will enter inside the cell and quickly the pores are sealed those cells will use for plating so this is all about the transformation of the E coli cells and I am sure you could have got the better practical experience of how to prep how to perform the competent cells and how to you know perform the transformations now let's move on to the next step and the next step is transformation in the East right so East is also another host which you can use for the over expression purpose and there are many methods which are available in the East the worst weather is lead acetate single standard DNA Peg method so in this method the yeast cells are incubated with a transformation mixture of the lithium acetate Peg 3500 peguins polyethylene glycol pack okay so tag is polyethylene glycol polyethylene glycon okay ah single standard carrier DNA and the foreign DNA or the foreign plasmid that is the recombinant DNA at 42 degree Celsius for 40 minutes the purpose of adding the carrier DNA is to block the non-specific site on the cell wall and made the plasmid available for the uptake Force transformation the cells are related to remove the transformation mixture and resuspended in one mL of water it is plated onto a solid media with the appropriate selection pressures such as antibiotics the second method is called as the experoplast transformation method okay and if video plus transformation method has multiple steps so in this method the East cell wall is removed partially to produce the spherioplast and that you are going to do with the help of the enzyme which is called asymolase so Speedo plus are very fragile for Oxford shock but are take up the free DNA at a very high rate in addition the polyethylene glycol pack is used to facilitate the deposition of the plasmid and the carrier DNA on the cell wall for the easier uptake so how you're going to perform the transformation of the yeast with the help of the Sphero plus transformation method so in the step one in the speed of last method the East cells are incubated with the zymolase the enzyme so first you are going to grow the cells at log face and then the step two you are going to incubate the cells with the enzyme which is called as xymolase so what the zymolase is going to do is it is actually going to chew up the cell wall at a very discrete step OK so you if you are not going to do a complete hydrolysis or cup complete removal of the cell wall you are going to make the partial removal of the cell wall to produce the speedo plus so the species or the cell what is going to be generated once you have removed the partial cell wall then it is called asperioplast ok now what you are going to do is you're going the step ah this video blast are going to be collected by the certification and incubated with the carrier DNA and the plasmid DNA for 10 minutes at room temperature so take the Sphero plus you collect the Sphero plus by centrification and then you are incubating the Sphero plus with the peg the carrier DNA and as well as the recombinant plasmids then in the step three it is now treated with the Peg and the calcium for 10 minutes with the gentle shaking so you are going to you know keep the cells like this video plus the carrier DNA and plasmid and then you are slowly slowly slowly you are going to add the Peg and as well as the calcium the transform this video class are plated onto a selective solid media and incubated onto the 30 degree Celsius for 10-4 days because it it takes that much amount of time for getting the Transformations so once you have got this you know transformed this video plus you are going to Plate them onto a selective media and then you incubate that into a 30 degree celsius incubator remember that the yeast is growing optimally in 30 degree compared to the 37 degree celsius and then you incubate that four days after four days you are going to get the plate with colonies and all these colonies are resistance for that particular antibiotic so uh this is all about these ah the methods the transformation methods what you can use to deliver the DNA into the bacteria or the yeast ah in this particular lecture what we have discussed we have discussed about how you can be able to use the different chemical agents to prepare the ah you know chemically competent cells and how you can be able to use them for T transforming the term with the transformations ah apart from that we have also discussed about the electroporations and so with this I would like to conclude our lecture here in our subsequent lecture we are going to discuss about the uh DNA delivery into the mammalian cells thank you foreign [Music] foreign [Music]