Hello, this is Joseph Guzzeri with MC freshman biology and today we're going to discuss genetic transformation of E. coli by the P. gloplasmid. This is experiment mini lecture for experiment 9. There's a few things that we need to know.
First of all, transformation. Transformation is a standard practice in which we change the DNA, we change the genetic makeup. Change genetic makeup of an organism.
We're going to be doing that to E. coli. E. coli, that is a genus and species. It's Escherichia coli, the actual genus.
So it should be underlined. It's not italicized in any writing that you see or what you put down. But the E.
coli... It's a very good subject for a couple of reasons. First of all, it reproduces easily, actually quickly, which means it can reproduce probably a thousand times per day. Another thing is that it's small and cheap.
and available. They're all over the place. And this is an educational, academic type of E. coli. This is not Ebola.
However, there are some types of E. coli that can produce stomach problems or other sicknesses. But this E.
coli, it's easy to transform. That's the process. We're changing the organism's makeup.
Now E. coli, or just like any bacterium, Some have flagella, flagellum. Some do not.
They are prokaryotes, which means they do not have a nucleus. They have a bunch of genetic material, a little area where it's found, which is called the nucleoid. But they also have, I'm going to put this in green, Little circular pieces inside them. These little circular pieces are called plasmids. A plasmid is a small ring of DNA.
It's extra-chromosomal. which means it's not part of the nucleoid. It's just normal genetic material. It's separate from the other bacterial chromosomes.
Bacteria can pass these plasmids to one another, either through asexual reproduction. Some can actually inject plasmids into others. Some can die and others come behind it and pick it up. But these plasmids can be passed around. These plasmids are also self-replicating.
So now we know what a plasmid is, but the P-glo plasmid is a very specific type of plasmid, which we will get to. How are we going to do that? Well we need to be aware of what we're working with.
A few chemicals that you should be aware of and familiar with. We got LB, we got AMP, ARA. LB refers to Luria or Tanny. L'Oreal Britannia is the food we're going to give E. coli for it to grow.
You're going to have two types. You're going to have a liquid form, or broth, liquid. This is going to be the extra food that we're going to give the organisms.
And we have some in auger trays that you will be using. So some nutrients are already in the trays. Some you're going to be giving extra.
AMP refers to ampicillin. Anytime you hear cilin, that's referring to an antibiotic. Antibiotic is referring to having bacteria.
Antibiotic means it kills bacteria. That's what antibiotics do. Now this kills, it's a very strong antibiotic, kills normal bacteria. We're getting that. So what we, hopefully you got an idea.
So what you're going to see is that ampicillin is going to kill a lot of our bacteria, but we're going to give it a special plasmid because they can easily take them up. We're going to make some E. coli resistant to ampicillin.
ARA is arabinose. Just from that last suffix there, O-S-E, we should see that it is a sugar for food if it has the enzyme to break it down, B-D, break down. Because of a lot of sugars, we can't break down unless we have the appropriate enzyme.
Where does the enzyme come from? Well, the enzyme, or the protein, comes from if you have the correct DNA. So we're going to change the DNA to produce some enzyme to help kill off or help it survive and be resistant to ampicillin. And then, let's talk about the actual specific plasmid that we're going to be working with. We've got the PGLO plasmid.
So this is a trademark plasmid with two genes. Two specific genes in question. OK, we bought this.
That's been put together. So here's our plasmid in question. We have a little ARAC.
We have a little section called GFP. And then we got. Gene BLA. What's new to this is the GFP and the BLA. Gene number one, BLA, stands for, refers to, Beta-lactamase.
Just from that name, you should understand that it is an enzyme. What it's going to do, it's going to destroy... If this DNA produces an enzyme, it will destroy AMP.
It will destroy that antibiotic. So if this plasmid is inside E. coli, it can survive on ampicillin.
It can just survive on a poison. The other gene, the GFP gene, refers to green fluorescence, spelling, green fluorescent protein. Maybe I should look that up. Okay.
Flower. OK. Green fluorescent protein. What it does is with a high frequency light, kind of like a black light, we can say, It'll cause, well it'll glow, it'll be fluorescent.
Fluorescent means if the light is being reflected off of it, it will shine more off. It's fluorescence. It will look brighter to us.
So this is not helpful to an organism. It's a Just cool. And there's a lot of organisms, if you Google GFP, that have this. They've done it in fish. We're going to do it in E.
coli. They have done it in pigs. They've done it in rabbits, rats, and so on. So what's the issue? How are we going to do that?
How are we going to get that plasmid into the E. coli? Well, we have to make the E.
coli competent. Competent means it needs to be able to do its job. It needs to be able... To receive the plasmid, we've got to get it ready.
We've got to be able to take it and put it in the mood. We're going to do that by chemical means and physical means. Chemical means. means and by physical means.
The chemical means is the calcium chloride, which is, I think in your lab manual, called the transformation solution. And so we're going to introduce that to it. Physical means is going to be by heat shock.
And some of these plasmids will be picked up. Or excuse me, the plasmids will be picked up by some of the E. coli, not all of them. But we'll see the ones that survive and do.
So what's going to allow that? What's going to actually, after we make it competent, after we get the gene inside there, how are we going to make this organism actually produce the protein from this gene or this gene? Well, we have to put something in the body to help induce it.
The inducer is what's going to activate the gene to produce the protein. For example, what's going to get the beta-lactamase to produce its gene? Well, what does it act on?
If it acts on AMP, we need to have AMP in the system for beta-lactamase. So ampicillin will activate. or induce, we could say, the beta-lactamase gene.
Well, what's going to induce, what's going to tell this DNA, what's going to tell this organism to produce this protein, the GFP? Well, that one's a little different. And that's going to be our arabinose.
Arabinose will activate the GFP gene. How this works is it's kind of like your body won't produce lactase. It won't produce the enzyme to break down milk if you never had lactose milk. How this works is this company that produced this plasmid. There's this gene on the plasmid as we see the ARA.
Alright, stands for Aravenose. Excuse me. It was for the enzyme Aravenase. Aravenase.
We'll break down Aravenose. That's what it was originally on here. At the beginning of every gene, you got this little promoter, it's like a little flag.
So when arabinose, the actual sugar that that acts on, is in the system, it's going to tell the body to produce that protein. Arabinose will promote Arabinase, usually. But in this case, this company came, left the very first part of that, cut the rest out, and then added another protein, excuse me, gene. So when arabinose comes into this system, the organism thinks it's making arabinase.
But in fact, it's just the promoter there. It's going to make the green fluorescent protein, only if that arabinose is there. And today, your job is to observe that.
You're going to observe these ideas in this lab. All right. So for Experiment 9, this is Joseph Gazzeri signing off.