Bacterial Transformation

hi everybody this is aaron from an elearning center i hope everybody's having a wonderful day i'm here to teach you a genetic engineering experiment so welcome what we'll be doing is we'll be working with living bacterial cells and we'll be introducing foreign or new DNA into these cells the goal of the experiment is to see if we keep these cells new DNA will they have a new trait so let's get started I said we were going to do a type of genetic engineering does anybody know why genetic engineering needs in general we think about genetic engineering its any sort of manipulation or change of DNA this can involve us cutting DNA in specific regions it can involve doing fragments of DNA together it can also involve transferring DNA into cells which again is what I'll be doing with you so let's go to the fourth through second I'd like to show you a little bit about DNA and bacterial cells so on our board here I have an image of a fragment or a piece of CMS does anybody know what the letters DNA scan for let me take out my pen dNA stands for deoxyribonucleic acid DNA is a molecule or a chemical that we find themselves DNA carries information in it does anybody know what the information in the DNA is for I think I'm going to draw my own strength of DNA on the cord so let me get a new page my image may not be advisable when I had before but you'll get the general idea as I'm drawing the shape of DNA does anyone know what the name of this shape is let me show you my bottle is back so right here at a beautiful model of DNA has so many different colors what's the name of this shape well we see some people answering excellent thank you so much this right here is a double helix and if I am twisted when exquisite like this dimples well in my opinion kind of like a twisted ladder or twisted staircase and if the eyes wasted we can see that it looks like a regular ladder or even so my students think it looks like railroad tracks we have not always known what the shape of DNA has been there were scientists working for many years to figure out with the shape of this molecule is and in 1953 dr. Watson and dr. Craig revealed to the world that DNA was in a double helix shape this is considered by many scientists have you one of the most important discoveries in the history of biology now why do you think it's important to understand the shape of a DNA molecule well when we're trying to study something it's so important for us to try to envision what that is in our minds if we can't actually see it and I work with DNA on the left every day but most times I'm looking at liquid that just looks like water I can't see the DNA itself because it's just too small it's important for scientists like me to be able to think about DNA and what it looks like so that I can consider how cells use or access the information in theater let's go back to the board for a minute because I want to talk about that information so here's my double helix on the board I'm going to highlight a specific region of the DNA right here this area that I've highlighted I'm going to refer to as a gene has everybody heard of a chain before genes are sections of DNA that carry information and that information can be used to make mRNA or proteins proteins are incredible molecules let me shrink up my information here for a second just so we have more room to write there we go so proteins the reason we're going to focus on proteins today it's because proteins have the ability to impact or influence various traits within organisms now has everybody heard of a trait before you can also use the word characteristic if you're more comfortable with that but let's consider different traits that humans can have or maybe even other organisms so a lot of times when I asked students to give me examples of creates some of the first traits that people volunteer tend to be things like hair color or eye color or height and though those are all fabulous examples you can also consider how traits are influencing how our bodies work so that can include how we digest foods or even though other molecules interact together within our body today we're going to be talking about a protein that's not as human protein the protein of our interest today is actually a jellyfish routine so I'm going to just give you one more slide here let me try to draw a Tele fish to the best of my ability here's my little jellyfish and this species of jellyfish that I'm trying to draw right here is Larry Victoria or the Pacific jellyfish and this jellyfish has an unusual trait it has the ability to make a protein that it that allows it to glow green so amazing now do humans have the ability to go green what do you guys think as far as I know on the outside meat don't humans do not make this green protein if we were to study the jellyfish DNA we would find that within their DNA there isn't gene like you were just assessing that codes for that protein and that protein that I've been referring to it's called Chi FP which stands for green fluorescent protein and if the cell makes GFP the way that we know that that cell is taking that protein well what do you think how do we know if it cell is making GFP that's so well protein so what I would like to do is you guys today is I would like to take that jellyfish gene and transfer it into living bacterial cells if our experiment is a success how will we know well if we give the bacteria some time to grow and replicate so we can see that our bacterial cells should have the ability to glory just like the jellyfish does what we'll be doing I wanted to show you our bacterial cells we'll be working with these individual cells but we're going to have so many of them so let's see we don't have hundreds of thousands of bacteria that we're working with our goal is to successfully transfer that gene into their cells so we must consider the bacterial cell itself and how we can work with those do you guys love bacterial cells what do you think I realize that bacterial cells get kind of a bad reputation while there are some bacteria out there that can make us sick the majority of the bacteria and the world are harmless to us in fact did you guys know that you have bacterial cells in your body you have an incredible amount bacterial cells in your body let me draw up here just a general bacterial cell so I'll draw the outside within their cells here their DNA just kind of floats around in the middle and these little structures I'm drawing on these little but these are ribosomes can anybody tell me why ribosomes are so important ribosomes are where proteins are made themselves so although I have not drawn much in this bacterial cell remember here's their DNA DNA is a set of instructions to make various proteins and bacterial cells have those instructions and they have the machinery necessary to build those proteins in addition some material cells have extra DNA which I'll draw over here this is a circular piece of DNA which we call a plasmid and I'll write that right down here for you and plasmids we shrink that up exist in some bacterial cells and what these cells can do with these plasmids if they can clone them which means they can copy those plasmids they can even share those plasmids if they want with other bacterial cells as scientists we're going to take advantage of these plasmids we take these plasmids so let me take this plasmid and I'll put it on another page for us let's see if I can paste it here I can figure that out that'll work look at that great okay let me just make it bigger so we can talk about it so here's our bacterial plasmid if you remember in the beginning of the lesson I said that scientists have the ability to cut DNA and DNA together as well so what we could do with this closet is we could cut a plasmid open and then you can also add in whatever our team of interest is so what that's not very pretty looking there but what we could add into this space it's becoming clear in our jellyfish gene our GFP gene in this experiment we also have a second team in our classes so let me just add that in on the right side so this second key I'll just draw it in a different color in reality it would not be a different color I'm just making that so it's easier for us to see this gene on the other side of the plasmid it's for something called ampicillin resistance that seeks me awhile to write a second or maybe a medicine that sounds like your facility what do you guys think ampicillin a lot of people realize it sounds very similar to penicillin and both ampicillin and penicillin are types of antibiotics antibiotics are medicines that are used to either kill or inhibit the growth of bacteria cells so if you guys have ever been sick with bacteria it's possible that when you went to your doctor they prescribed to you an antibiotic to get rid of that bacteria that was making you sick in the lab we can use antibiotics to help us with these experiments what I have over here this gene is not a gene to make that antibiotic this is actually a gene to enable bacteria cells to resist that inside the attic tell me what it means if bacterial cells are able to risk in antibiotic it means that these bacterial cells will not be destroyed or even affected by that antibiotic if they have the ability to resist it you might be wondering why would we be making bacteria glow and giving them the ability to resist an antibiotic well let me show you just why this gene is so important for this experiment let me draw my little bacterium cells again for you so there it's got my cell with its DNA and it's ribosome I didn't need to do that let's give it a little more DNA there hold on we put that together and I'm just going to rotate that a little bit and let me make lots more of these cells so in our experiment we're going to have so many bacterial cells our goal is to take these plasmids remember what we were just discussing I want to take that plasmid and I want to get it into some of these bacterial cells I don't have to get it into all of the bacterial cells but I do want to get it into something so let's draw it in blue let's say I successfully transferred the plasmid into this cell right here if the cells take up that plasmid they'll have the ability to make TSP which is great if they're making shifting they should also be blowing great awesome the problem here is that if I only had the gene for GFP in that plasmid you see these other cells out here that did not take up the plasmid we call those untransformed cells those untransformed cells what they would do if they don't take up the plastic they're just going to grow right on top of that trans well the one that did take up the class and I'm never going to see that cell so that would be a problem in order for me to find the bacteria that successfully take up that plasmid I have to eliminate these other cells somehow and that's where that second chain comes in that resistance gene well if my bacteria so with the plasmid also has that antibiotic resistance gene think about what would happen if I add ampicillin to this environment right now the ampicillin would inhibit or block the growth of these other cells so let me just take those off the screen but the ampicillin would not be able to affect the cell that took up the plasmid so this resistance gene is so important because it allows us to isolate the cells that we have transformed that we keep the DNA here so if you guys are ready I'd like to start and show you the different steps of the lab so in front of me I have a lot of equipment if you guys have worked with us before maybe you have used some of this equipment one of my favorite pieces of equipment in the lab is a pipette I love using pipettes these are used for measurement they're used to measure small volumes which we call microliters and the symbol for microliter looks like this there are 1000 microliters in 1 million and what I'd like to do right now on my iPad is I'd like to start off by setting my hat on 250 microliters I'm going to just adjust the dials so that I don't know if you guys can see this but I said it it says 0 to 5 0 which is 250 I'm going to also show you that there are tips that go along with these I so here right here I have a box of tips or bottoms that I can change throughout the experiment and keep everything sterile do you guys know what it means if everything is sterile in the left if we're keeping everything clean or keeping it free of contamination and I'll just show you for practice what it looks like when I place one of these tips on so I've got my bed just gonna place it on and you see how it just attached itself I can then use the pipette to transfer liquid between tubes and when I'm done I'm just going to change that together and get a new one if I mean all right so I would also like to label some tubes so that everything is prepared from once you start we're going to be working with two tubes in the beginning and if you guys are using our worksheets for this lab try to take as many notes as you can I always like to keep a notebook and write down all the volumes and the different reagents or materials that I add to each soon so for our purposes we're gonna have a plus 2 and then minus 2 and before they even write what I'm going to add to those tubes I'm actually going to label the right in front of you so I have two empty tubes here these are 1.5 milliliter tubes that we use I'm going to place a plus on one of you just like that and then we put that I like to use a rack also to keep everything organized and then I'll be placing just a simple - on the other two to see that but that one in the rack as well we'll be transferring 250 microliters of a liquid called calcium chloride so this is our calcium chloride here this is just a special saltwater solution that we use and remember I said I'd like to keep track of what I'm doing so I'm going to add that add a 250 Mike of calcium fluoride that - and oh my gosh like just erase everything they did let me start again 250 microliters calcium fluoride I guess I'll just make it over here 250 microliters calcium chloride alright let me show you how I do this with the pipette so I'm going to open it tip box and I have my calcium chloride just double-checking that my fake head is correctly set open the calcium fluoride maybe you guys can see me pipetting gonna go to the first stop on this pipette gently please will let the tip up a pipette and it's a little liquid and I'm slowly going to drop if you guys notice I'm keeping everything at eye level so that I can see exactly what's happening I'm gonna pick up one of my suits place the tip toward the center and just gently release all that liquid keep that tube closed and when I added the calcium for it I'm now going to place that on ice all right next to same thing look at my calcium chloride one of the first stop gently place it in the liquid and slowly drop no air bubbles well sorry about that let me get my - - great when a close that's even also put it on ice and I'm gonna change the test what I'd like to do now is I'm going to add the bacterial cells that we'll be using in this lab we're using a type of e.coli bacteria called mm - 9 4 these are harmless bacterial cells and they're willing to take up the DNA network you want to introduce into them so they're perfect for this laughs have you guys heard of e.coli before there are some strains of E coli that can be a harmful or dangerous but this is not one of those strengths I want to add it to my board that I'm adding the bacteria as well so mm 294 it's gonna go in both of these columns or to distinguish tonight back let me show you bacteria so we grow a lot of bacteria in the lab all the time here and if you could take a look at this plate let's see I don't know if you can see the but there is material all struck on this plate let me show you an empty plate real quick so you can see the difference all right so this one over here is just empty it has food for bacteria in it and this one right here has the bacteria struck on top of that food so they're just crawling on top of that food and my job right now is going to be to Sara Lee transfer the cells from that cage radish into the tubes that we just put the calcium chloride into first of all I'm going to be using heat i'm going to be using flame so since i have long hair i'm just going to pull my hair back to be safe and I'm gonna get my burner out nice it's just gonna be gas I'm gonna turn the gas on and I'm gonna light it this spark there you go just a second all right all right now I do wait to work close to those lab because this area right here it's considered a zone of sterility what I mean by that is that because of the heat I don't want to work so close to the flame that I get hurt or that it is just too hot in general but I'm gonna work close enough this right here you guys see this this is what we call an inoculating loop and what I'm going to do you're certain activity we can see a little better against my shirt there okay the inoculating loop is literally just a piece of metal and I'll drop this very simple word like this which has this circle and I use it as a method of just scraping up or lifting the bacteria's house so first what I want to do is I want to sterilize this lip because it's been sitting in the lab and it's just not clean so first of all places in the flame and when it's orange like that it should be clean it's also super hot when it's orange so if I take this flaming hot piece of metal and I pick up living bacterial cells what do you guys think might happen to them I bet that if I touched them with this really hot piece of metal those bacterial cells are gonna die so I would like to before I take the bacteria up I'm going to take this loop and just press it into the gel in the petri dish to cool it off the loop should still be sterile when I do that you'll notice that I'm holding the plate doing my best to try to keep anything from contaminating the plate and I'm just scraping and lifting in a small direction with this loop I'm trying to get the bacteria off the dish just like that let's see if you guys can see that all the lid up against my shirt I don't know if you can see that you'll get a darker piece of paper to show you guys if I hold it against this yes yeah it's a little bit kind of like a clump of these or white right there yeah those are the bacterial cells it may not look like a lot but really there are so many cells in that box oh no but what I might use I don't want is fine and I'm gonna take that and just please get right into the calcium chloride I'm swirling the bacteria off the loop and sticking so this part can be a little bit frustrating there we go okay and if you take a look at that it's like using this so you can see it filled the liquid is now cloudy and that's an indication to me that there are a lot of bacterial cells there if you're able to see that it doesn't like Europe much closer bacteria which will take care of in a second let me repeat that for the next tube so remember I want to sterilize my loop nice and clean I'm going to pull that loop off so I don't kill the bacterial cells right into the gel perfect and now I'm going to try to sweep and lift if I tear you off the dish great let me pick up my other two men up holding everything's eye level again they're being a little city today they're sticking to the loops a little bit harder to get them off almost there excellent close that too but in the ice and I'm just going to clean the one more time you guys may have noticed that I've been keeping the cells in ice and I'm going to explain that to you in a minute for this portion of the lab I like the cells to be nice and cold we're done with our flames I'm just going to move that aside and if you remember I said that there were some clumps of bacteria in here and I'd like to take care of those so you can use the pipette to just mix up the cells I call this three suspending the cells so I'll take a new tip I'll take either of my tubes I can start by the one and before I go a little like what I'll go to the first stop and I'm simply going to draw up that liquid and the clumps are getting stuck and release keeps doing that and this may take a few times but that's perfectly fine there we go that looks great I'm going to do the same thing for the other two just drawing up and releasing if I have any bacteria on this side it could use it you just if I had to just kind of spray that bacteria down or wash it back into solution alright now at this point what I'd like to do is I would like to add our class Vidya we're going to add the plasmid to our plus tube only and that's really what the plus indicates to me the plus is plus plasmid the minus is the minus plus I know with oh my gosh I'm going to every slip on board here let's try that again there we go this group here we know I'm adding ten microliters of the plasmid that contains the GFP gene so what we did is write it before on that plasmid just show you this plasmids name is PG SP the plasma today's so the lowercase P central plasmid and the GFP is an indication of that gene of interest rate so we'll come back perfect alright so since I'm using only 10 microliters the blue make that would not be appropriate the blue pipette only measures from 100 to 1000 microliters this yellow pipette which I'm not using now we will use at the MS measures from 10 to 100 microliters and this clean one here this is the one that measures from 0.5 to 10 microliters so I'll be using the grey one for introducing the plasmid DNA into the PAS students the gray pipette has its own tips and you'll see just how small they are in comparison let me get my plasmon have the plasmid in this green tube here so the plastic that were working with is in a concentration of point zero zero eight micrograms per microliter which I'll add to the boards you have for your notes that just has to do with the concentration of the DNA is telling me essentially how much DNA is in that liquid if you look at the bottom up and scoop everybody whenever I get these tubes that's my since they only say Erin there is nothing in my to take a look there's a little baby bit of liquid a lot of all most decisive is your job it's going to be plenty for this experiment so let me use my paper my five let me bring it up to ten all right I'm going to draw up and using my eyes I want to see the liquid going up through the tip when a notes are sure that I'm taking that liquid up and it was plenty in there let me get my plus two when we add the plasmid I don't want to add it to the top of the tooth I wanted to go directly into that cell mixture and then I'll release right back to the ice because we have been working for a little bit my ice is getting a little bit melty but that's perfectly fine I'm going to go into why the ice is so important and how will be changing temperature and v just now so right now I want to draw what's in our plus two I'm joined by bacterial cells here with some openings in that membrane at a certain point in their development these bacterial cells can have these openings which we call it Asian zones or zones the division and there's my bacterial cell let me make some more cells we're going to use those in fusion zone to our advantage alright so if you have a bunch of bacteria if this were the plus to remember what did we just add to the plus two we just added the plasmid DNA to the mustard let me just draw some plastics I'll just make them blue so we've got a whole bunch of flashes in there now at weights line I have the plasmids on the outside of the cells if we want these cells to use this DNA and to make these proteins where do you think we want these plasmids to be if you said we want them to go into the saltier absolutely right you want a method of pushing the plasmids into those cells there are a few considerations can't cure them so first let's think about the DNA molecule itself so DNA does carry a charge on it and I'll look right here at my molecule okay so we've talked a little bit about the beginning let me just let this in here okay so I love that this model has different colors on it we've got the white and the black on this side and that's our sugar phosphate backbone and we've got our nitrogenous bases in the center's so this is really the code of DNA is in the center these are our ace T's CS and G's well we were discussing the charge that's on the DNA I'm interested in these phosphate groups out here the phosphate groups on the DNA molecule have a negative charge so when we're working with DNA you want to remember that DNA carries that negative charge if we're trying to push the DNA or a negatively charged molecule into a cell let's assume that membrane as well so the membrane has a negative charge just like the DNA molecule and that has to do with the phospholipids in there the DNA has a negative charge and the membrane has a negative charge does anybody know why that might be a problem for us if we're interested in pushing the DNA into the sex well well what happened to the DNA is that the DNA would be propelled or pushed back because of those two negative charges so what do we need to add to this environment we need some positives the reason that we use the calcium chloride is to neutralize this environment so let's draw some positives on here once everything is neutralized I then still need a method of pushing the DNA into the cell and it's amazing we can use heat to push DNA into these cells he creates movement of molecules so remember that our cells are nice and cold right now and it not much is moving around in there because everything is cold but think about it if beats take these tubes which are cold now and we learn them up you should get some movement of those molecules and that includes the DNA I'd like to show you an animation that does a beautiful job of this next step for us let's see if I can pull that maybe not your denied happen biron it's perfect okay so this step is called the heat shock here's a beautiful picture of bacterial cells they did a much better job of drying the bacteria than I did and you see those nice and fusion zones right there what we'll do is little soup on one of those occasions okis perfect so this area in the center is that opening that I was trying to draw and then we have those possible in between fans around all over the place in the membrane you can see that they have their negative charge as well if at this point we try to add a condiment to this cell what I predict would happen is because the membrane is negatively charged and the positive is negatively charged I predicted that positively get bounced out and that's what we're going to show you right here oh did you see that Osmond come in and then get pushed right out like that so in order for this to work for us we're going to take a few steps here we're using the calcium chloride and we're going to put the like what I'm gonna sell em I use well barium calcium fluoride watch what happens everything slows down and everything is now ready for us to transfer that DNA into the cell if you remember all we need is a little bit of heat at this point been a number of experiments that have identified that the best temperature for transferring the DNA is 42 degrees Celsius so watch what happens when you raise the temperature at this way to 42 degrees Celsius we expect the tenant get pushed right in in order to encourage that DNA now to study in that cell I would want everything to slow down so I'll place everything back on ice I'll draw many steps out for you just select nice and clear and again if you're taking notes in the lab you can write this down so here's our neat shop and it's a set of three temperature stages so the first one works point right now we're doing the ice anybody know what the temperature of ice is in degree Celsius zero degrees Celsius so I like to do that step for about 15 minutes and we're doing that right now so they'll just put a little check mark next step in the next step I want to bring this temperature up to 42 degrees Celsius and I realize that the work in Celsius in the left so if you're more comfortable with right this is approximately a hundred and eight degrees a lot of people think that's a very high temperature I suspect that I won't think about your body temperature body temperature an average temperature would be about 98.6 degrees Fahrenheit so this is just a little bit warmer than our body temperature you would never want to have a fever that high but I think you've taken a shower or a bet that is about that temperature we'd like to do this temperature for 90 seconds once we do that step I hope that some plasmids have been transferred into the cell and to encourage them to stay in there I'm going to play some on us I'd like to do that threat least 1 minute so you can do that under but we definitely want to do it for at least a minute so let me show you not simple to each obvious I have a water bath over here which is set to 42 degrees Celsius nice cool month and I always make my Beach out distinct so my tubes are either in the ice or they're in this water bath and they're nowhere else so if I were about to start my heat shock I would get my timer set I'm going to take both tubes out because both needs it through this step and I place them right here in this rack in that water and after 90 seconds so we can pretend that 90 seconds went by I'm gonna take those tubes and I have my beaker right next to me I'm gonna place them right back in my X and again it's okay my - my ice is getting a little watery no problem but I'll leave those in there so what I expect has happened is the cosmic temple moved into some of those cells one more step I'd like to take is I would like to give the bacterial cells some liquid food so be using my iPad again it happens to be on 250 microliters so let me add that we go back to our little slide here perfect let me just add this step because I don't want to forget I'm going to add 250 microliters of what we call LV which is the bacterial liquid food that's gonna DeLong constitutes it really so if you're looking at this chart it displays nicely that the only difference here is this is that we added the DNA of us to get up to the minus two all right so let's add our own be alright just check up so on to 50 get my healthy out over here I'm gonna draw that up get I have to make an assault with anyone that doesn't matter I'm gonna add that to the to change my scale get anyone now they're 250 from the LP at this point I can discard this just as my next two I'd like to know if everything worked so in order to do that I have to take those bacterial cells from the tubes and grow them on petri dishes I'm going to show you the petri dishes that I have here is already labeled them okay so they look just like this and all I do is I just take a sharpie marker and I write it on the bottom of the dish the information that I need I'm just going to display on the board free because I know it may not be clear when you're looking at my dishes like that the information that I have we have it somewhere down there so thanks I have four petri dishes for this lab let's see if I can highlight this so over here this is just a little key over here so that's stable line it represents a petri dish that just has lb of food in it that double line right there is a petri dish that has ampicillin in it as well so I am using two different types of petri dishes in this labs you'll see up here these right here are my 2 lb plates and these down here are my 2 ampicillin plates and also remember that we have two different tubes as well we have plus tubes and we have - tubes so we can get a little confusing and I always like to keep notes on how I label my plates I am going to take the plus bacteria so bacteria from the plus tube and I'll be adding it to any plate and we just make this look more clear so there's a plus that has a plus sign this is also a plus and my - bacteria just draw angry movements little easier okay I'm gonna have the minus X here it to any plate that has a minus so if you can imagine let's say this is my - - this is my question what I'll do in a second you'll watch me is that this going to take 100 microliters from these tubes and plate it asks each of those please it's not very mean I apologize for my handwriting all right if I were just to add bacteria to the center of the plate they would just be sitting in this little puddle which would not be very useful for us there are different methods of spreading bacteria so I'll show you the one that we happen to like to use in this lab so I'm going to take over here they have these tubes which have little sterile class beads in there to eat your mouth this is little beach in there what I want to do is put one of these tubes on each of these plates so I'm just going to open it to do my best just to dump the beads on there what often happens when I do this is that some of the beads bounce out on the table or do you think it would be a good idea let's say I dump the beats out and they fell on the table do you think it would be a good idea for me to pick those beads up and throw them back into the petri dish I definitely would not wanna do that I would not want to do that because Beach once they're on these people they wouldn't be sterile and if I place them into the dish they could contaminate the experiment so I would always just get a new tube of beads if that were the case all right so I'm ready to plate my bacteria if you remember they said it was going to plate a hundred microliters so yellow playhead is what I'm using for this on the brandy yellow pipettes we have lines that represent the smooth ways so this is a hundred microliters I'd like to get organized so among a few traditions remember I have all my labels going I'm just going to separate out these are my US dishes and these are my - so I'll take my plus to feel a mess up I'm going to take out a hundred and I'm just going to let them clean up a little bit and release the liquid automatically doing exactly the same thing from the same two for the second plus plate and then I'll discard that and change my tip same thing for the - sticking out I see old my face if you were working with a partner and you wanted to share the work you could so that clinking sound that you hear is the beads moving around as I shake the place and what the beads are doing is as they roll across the surface of the plate they are pushing and moving the bacteria evenly across the surface I like to do that usually for about a minute to make sure everything is evenly spread and once I've done that I do the beads very simply by holding both sides of the dish because at this point I would take my petri dishes and I would flip them so they're upside down and that would place let's pretend I'm placing them in an incubator overnight at 37 degrees Celsius when I come in in the morning I should see if everything is work I should see some bacterial growth if you don't have an incubator which many of us don't you can leave the dishes out at room temperature they just take a little bit longer to grow because they're usually at a colder temperature they're at room temperature I'd like to show you just when it looks like what the expected results are on the floor and then I'll show you five are real plates okay so let's consider this if a patriot just has scoops of just as lb so this one right here is what I'm talking about okay and I add bacteria from the - to understanding regular bacteria and calcium chloride - a plate with food what would you expect imperium cells like that let's try it again we'll do it this way the bacterial cells will rub all over the plate that kind of a growth where you just have the bacteria everywhere that is considered along so let's say I take those same cells okay so you put the stem cells and I'm going to add them to this plate right here oh I didn't want to do that I'm going to add them to this plate right here this one those are regular bacterial cells and I'm adding them to a petri dish that has the antibiotic ampicillin ah what do you think is going to happen if that's the case so you're introducing bacteria into an antibiotic their growth is going to be inhibited so I would expect there to be no rows on this plate that is an important way for us that is telling us that our bacterial cells are inhibited by the end the scylla and it's also telling us that our imbecile and it's working at the left now let's consider our plus plates if we're experiment is a success remember we should have been able to get some of those plasmids into some of the cells in the plus 2 I don't expect that all the cells in the plus 2 took up the plasmid but that's fine if those cells took up the plasmid I would expect them to have the ability to flow 3 so there should be some glowing bacteria on that day but keep in mind that the bacteria cells in that Lane I'm sorry in that tube all of them did not take up the plasmid so we would end up with we try this one more time bacterial cells there we go that don't take up the plasmid and grow on top of our glowy cells so although these two petri dishes right popular with everything is bouncing around here although these two petri dishes right here these two right here well look the same must they are genetically different now let's take a look at this point right here so this is a plate where i'm adding the same cells from the plus two I should get glowing cells on there if everything has worked and I am adding to this plate remember I am adding still those cells that did not end of the class but think about this yes we hear this theme puppet keep Zuma this petri dish okay this one right here it has the informatics implement and we said that facility is going to inhibit the growth of any bacteria that don't have that fast so we're gonna get rid of those the only cells that were hopefully gonna be able to see on this plate are going to be the glowing cells I'd like to show you because our plates aren't ready yet because we just did this I'd like to show you a petri dish that I have from already doing this experiment the bacterial cells that glow green they do have a green color to them but when we put them under a UV light the GFP protein grows I'm sorry glows super bright so if we can I'll see if my friends can help me here we can kind of turn down the lights I'll put a black light on and you can see you guys still see me here right in the camera side so much what do you think so here we go look at this oh they're so beautiful oh my gosh can you guys see this and click over there yeah here are glowing bacterial cells and we can see that they have that jellyfish protein they're making that jellyfish protein this plate that I have in front of you guys is it has a lot of bacteria cells on it let me show you one that just says less it's still an absolutely beautiful plate and before we finish up I just want to explain to you I'm not going to hold up this one which has fewer cells here okay you can see that there in these little dots do you guys see that so those individuals dots we call those colonies so each of these little colonies here that is the result of a successfully single successfully transformed cell so that cell took up the plasmid survived on the plate and reproduce to replicate it and now we have a whole clump bacteria in that region so each of those little colonies should have clones of bacteria all around it so I hope you guys had a good time today if possible I'll see if I can be be cancerous no questions and I wanted to let you know that we do have worksheets available for you on our website and that will be continuing into a variety of different experiments right with you guys so hopefully you can check back every day and if you're available you can keep working with us let's see well I just want to say thank you that everybody for all your hard work and for giving your attention I'm so grateful and I hope everybody has a wonderful day

Channel: DNA Learning Center

Views: 2,139

Rating: 4.8297873 out of 5

Keywords: e coli bacteria, dna transformation, dna molecule, herbert boyer, stanley cohen, recombinant dna, dna sequence, e coli, plasmid, expression, Bacterial Transformation, David Micklos, DNA Learning Center, Erin McKechnie, Cold Spring Harbor Laboratory, GFP, green fluorescent protein, protein production, genetic engineering, ampicillin, antibiotic resistance

Id: jemSJSKXh4U

Length: 54min 33sec (3273 seconds)

Published: Wed Mar 25 2020