Plasmid transformation

hello my name is ed Chapman and this videocast is going to explain how to use bacterial plasmids to deliver a gene of interest into a living bacterial cell and cause that bacterial cell to transform to acquire abilities it didn't have before whatever abilities it is that that gene of interest that you picked will give to it so in order to do this we're going to need a basic list of supplies we're going to need some need some living bacteria and in this case we're going to pick ecoli bacteria that are negative for a gene called Lac z this means that these ecoli bacteria do not have the ability to digest lactose we're also going to need some agar plates to grow the bacteria on now these plates are going to contain the basic nutrient media which basically contains all the vitamins and minerals plus glucose that bacteria need to grow plus we're going to add two special ingredients we're going to add the antibiotic ampicillin which normally kills bacteria and we're going to add a an extra sugar called X galactose which is a product that when the bacteria digest it it produces a blue metabolite and this is also known as X gal we're going to need our gene of interest from the heat from a human for example the insulin gene from humans and we're going to need a restriction enzyme that can cut both the plasmid and the gene of interest and we're going to need our plasmid and then in this case we're going to use a plasmid that contains an ampicillin resistance gene and a gene that gives the ability to digest lactose called Lac Z so the plasmid is carrying two genes let's use this diagram here to map or chart our progress through this procedure so here up here in the upper left we have our bacterial plasmid remember plasmids are loops of DNA that contain genetic material that's separate from the main loop of material in the bacterial cell this plasmid we have engineered it or select it so that so that it has the ampicillin resistance gene which is colored in orange right here and we also have the lac de Z gene which is colored in white right here now notice that the restriction site remember restriction sites are the locations where restriction enzymes can cut or break open DNA the restriction site is located right in the middle of the lac z gene so when we add the restriction enzyme it's going to break the plasmid open but only at this location so we are literally going to be cleaving the lac Z gene in half and whenever you break a gene open and insert new genetic code in the middle of it kind of like creating an artificial intron here you now render the gene useless the gene cannot work and so that's going to basically shut down the lack of z gene if we insert anything in this space okay so our first step is we're going to isolate or purify our bacterial plasmid and our gene of interest okay the gene of interest over here is from a piece of human DNA and the gene of interest is colored in black and it's got some other stuff that needs to be cut away by the restriction enzyme okay so what we're going to do is we're going to take our plasmid and our gene of interest and we are going to cut both of them with the same restriction enzyme so what this is going to give us is plasmids that have been opened up because they've been cut at their restriction site and the human DNA is going to be cleaved also in two pieces and we're going to we have selected the restriction enzyme so that it will cut here at a known location and only one known location and it is there's there's restriction sites that are bracketing the gene of interest that we want so we don't want to use the restriction enzyme that's going to break our gene of interest that wouldn't make any sense at all so you can pick restriction enzymes for certain restriction sites that we know are not in our gene of interest so we mix the restriction enzyme the plasmid and our gene of interest together and what we end up with is a cut open plasmid and a gene of interest that's been cleaned up and we're going to mix them all together and we're going to add an enzyme called ligase remember DNA ligase has they go order to kind of patch DNA back together and if everything works we're going to end up with a plasmid that has the gene of interest inserted where we wanted to put it okay and ligases of course is going to seal the restriction sites close so now we have a complete functional plasmid okay and then we're going to take our plasmid which has the gene of interest inserted into it and we're going to mix it into a culture containing our ecoli bacteria all right now remember ecoli has its own DNA here in the loop called the nucleoid and if everything works and the e.coli takes up the plasmid it will now acquire the ability to resist ampicillin it will be ampicillin resistance resistant but it will not have the ability to digest x-gal because it's Lac Z gene is broken because that's where we have inserted the gene of interest so then we're going to put our bacteria that we have hopefully transformed onto a clean agar plate and we're gonna streak the plate which means you're gonna take your your bacterial plate and you're gonna rub it with um with the bacterial with the bacteria that you've attempted to transform so that you're spreading them out all over the plate and then you're going to incubate it and we're gonna see which ones grow okay now on our plate if everything works because remember it contains ampicillin we should get two different colors of colonies growing we're gonna get blue colonies and we're gonna get white colonies okay now the blue colonies are not the ones who are interested in because remember the blue colonies have taken up a plasmid that has just reattached so imagine it's this plasmid without the gene of interest in it so these two little white parts which are the Latin z gene has reattached to itself a Rhian ield so that happens we basically restore the plasma to what it was before and if a bacterial cell takes up that this plasmid without the gene of interest in it it's going to have the ability to resist ampicillin and grow but it's also going to have the ability to digest x-gal and produce the blue metabolites so those colonies are going to be blue the white colonies on the other hand are the ones were interested in the white colonies will have taken up this plasmid right with a broken lat Z gene but they still have ampicillin resistance so they can grow but they don't turn blue they turn white okay and then we're going to grow these bacteria by the trillions to produce lots and lots and lots of copies of this of this gene of interest that we have put in it alright so let's look at this slide and try to pull all this information together the bacterial cells that did not take up any plasmid at all are not going to grow because they're gonna be killed by the ampicillin so you're not gonna see any of these cells they're gonna they're not going to appear as visible colonies because they're dead the ampicillin kills them the bacterial colonies that grow blue okay the little blue specks you see on here these are millions and millions of bacteria that are cloning themselves from an original bacterial cell that took up a plasmid but the plasmid didn't take up it wasn't a transformed plasmid so the plasma does not contain the gene of interest it has a completely functional Lac Z gene so these guys are going to metabolize galactose and turn blue we don't want those so it's one of the colonies were interested in the colonies that took up the transform plasmid are going to grow white and those are the ones we want so the colonies on here that are white are the ones that have done a couple different things they've taken up the plasmid okay they have the plasmid and they also have the plasmid with the gene of interest inserted okay and when the gene of interest gets inserted into a plasmid we say that it is a transformed plasmid alright and that's what we want so then we're going to get our white colonies so what you then do is you take a bacterial loop which is sterilized in a Bunsen burner it so it's a basically a metal handle with a little loop of metal on the end of it sterilize it scoop out one of those white colonies and rub it onto a fresh agar plate alright and then you incubate that in which you should now get is lots and lots of little colonies of just the white bacteria and you can repeat this process to your heart's content and grow as many trillions of these as you want the best example I can think of an industry where this is something like this has happened is how genetic engineers created a drug called Humalog and Humalog is a human version of insulin that has been grown by bacteria so what you basically do is you transform a plasmid with the insulin gene and you insert that transform plasmid into a bacterial cell and then you grow these bacterial cells in fermentation tanks by the quadrillions and then you refine out of that bacterial soup the insulin that the bacteria are going to be translating from the messenger RNA that they transcribe from the DNA that you inserted which originally came from a human so you literally have created a franc and bacteria that does something that no bacteria can do it can make insulin which is of course an important hormone that humans that some people need to take to treat their diabetes and this this diabetic drug now is produced cheaply and cleanly and purely in the form that humans can tolerate the best which is human insulin just like your own body would naturally make if he did not have insulin so this is a great example of how a genetically modified organism in this case a bacterium has helped and probably save millions of lives over the last 30 or so years as long as this technique has been known thanks for listening and we will stop there

Channel: John Chapman

Views: 29,495

Rating: 4.9346404 out of 5

Keywords: plasmid, bacterial transformation, xgal, gene cloning

Id: fafwE5flnDw

Length: 10min 34sec (634 seconds)

Published: Mon Mar 02 2015