[Caitlin:] Getting ready for a special occasion? [Felicia:] What? You told me we were going to do transformations today. I've got everything we need for our make-overs! [Caitlin:] Urg, Felicia! Bacterial transformations! Not beauty transformations. [Felicia:] Oh, yeah. That makes a lot more sense. [Caitlin:] I've got heaps of cloning to do. Yesterday, I used the restriction enzymes to do the cutting. Now, I need an extra set of hands to help me with the ligations and the transformations. [Felicia:] Okay. This is great. I guess I can forego the manicure and put my hands to good use in the lab. Let's get to it on... [Both:] DNA Decoded. [Music] [Felicia:] I'm still on my quest for a blue tomato. Let's recap. Manipulating DNA involves three steps: Cut, Paste, Copy. [Caitlin:] So far, we've talked about how to select molecular scissors that will seek out palindromic DNA sequences and cut our gene of interest. In this case, the gene that makes blueberries blue. We're using two different molecular scissors, or restriction enzymes, so that we can make sticky ends at either end of our gene of interest, so it can only fit into the plasmid in the correct orientation. Remember, we've also used the same restriction enzymes to make cuts in circular plasmid DNA. As you can see, everything is lining up nicely. The sticky ends of our gene of interest are compatible with the sticky ends of the plasmid DNA. The bases will fit together according to Watson and Crick rules: A pairs with T and C pairs with G. [Felicia:] Now, we just need to glue the plasmid DNA and the gene together. [Caitlin:] Easy for you to say! But it's a bit more complicated than that. Remember back in week one, we said that the bonds between the base pairs are weak. At this point, our plasmid DNA is only held together by these weak non-covalent bonds between the base pairs that formed the rungs of our DNA ladder. To make or modified DNA fragments sturdier, we need to create strong covalent bonds between the phosphates that make up the sides of the DNA ladder. To do this, we'll need some molecular glue. Luckily, we have some: an enzyme called DNA ligase. DNA ligase joins the fragments of DNA together. It's the "paste" component of DNA cloning. This process is called ligation. [Felicia:] So far, we've been showing you a blobular version of ligation, but here you can see DNA ligase in action. In this image, the DNA ligase is in purple. You can see that it's folded and wrapped around a green molecule. If you look closely at the green molecule, you can see the familiar DNA double helix. What you're looking at here is DNA ligase binding-and-pasting two pieces of DNA together. [Caitlin:] Okay, back to our cloning project. We've snipped our gene of interest, inserted it into the plasmid DNA, and glued the ends of our plasmid together. It's probably a good idea to make copies of our plasmid. That way, if we want to use it in other experiments, we can. We could see if we make bananas blue, or oranges blue, or even dogs blue. [Felicia:] Caitlin... [Caitlin:] Ah, yeah? [Felicia:] How are we going to get through the cell wall to deliver our blueberry gene? [Caitlin:] Yikes! Yeah, there is that. [Felicia:] That's going to be quite a bit of a challenge. [Caitlin:] I've got it covered. Do you remember when I told you that restriction enzymes are like little ninjas that attacked invading bacteriophages? We learned how to harness their awesome swordsmanship and put them to use as molecular scissors. I hope you don't have a queasy stomach. [Felicia:] Um... No. Why? [Caitlin:] Because now, we're going to talk about how we can harness bacteria's ability to replicate like crazy to help us make lots and lots of copies of our plasmid. Meet E. coli. [Felicia:] E. coli? Seriously?! That's the stuff that gives you gastrointestinal infections. [Caitlin:] Yes. That's the one. There are many types of E. coli. The ones we use in the lab for cloning are completely harmless. The bacteria E. coli is one of the most common organisms used to express genes. Let's check out our little friend here. [Felicia:] Um, it kind of looks like a hairy drug capsule with a tail. [Caitlin:] The hairs are just there to help it move around. Just ignore them. [Felicia:] Okay, so we have E. coli and we have our plasmid DNA. But how do we get our DNA plasmid IINSIDE the E. coli? [Caitlin:] This is challenging because E. coli have several barriers that we need to get through. It's like a fortress surrounded by a fortress surrounded by another fortress. These barriers are designed to keep things from getting inside the cell. So, we need to weaken them -- at least temporarily. [Felicia:] Okay. We can either do that with chemicals or with electricity. We can use a special type of calcium to soften or increase the permeability of the E. coli cell wall and plasma membranes. That makes it easier for the DNA plasmid to leak right through into the cell itself. Or, we can give the cell an electric shock that punches temporary holes into these barriers to allow the DNA plasmid to sneak through. [Caitlin:] Okay, so now we're in! We've cut. We've glued. And now the plasmid DNA is inside the E. coli cell. The process of sneaking the plasmid DNA inside the E. coli is referred to as transformation. The E. coli has been transformed into a molecular-copying machine. Now, we can start to make lots and lots of copies. [Felicia:] Oh, and one final thing. Despite what our simplified diagrams show, we're not just sneaking one DNA plasmid inside a single E. coli cell. In reality, this process is much more complicated. We have skipped over some of the details, but this is the basic idea of DNA cloning. [Caitlin:] Armed with this arsenal of information, let's move on and talk about -- you know what's coming next -- [Felicia:] Oooh, mutations!
