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Biotech: Genetic Engineering 2,020 views

Teacher/Instructor Patrick Roisen
Patrick Roisen

M.Ed., Stanford University
Winner of multiple teaching awards

Patrick has been teaching AP Biology for 14 years and is the winner of multiple teaching awards.

[0:00:00]
I started off college as an art major. I only wanted to become a Bio major by accident when my girlfriend that I had at the time wanted somebody to sit next to her in Biochemistry. Well, you may be able to guess why. I started taking those classes, but what kept me in them and made me go from just being a minor in Biology, to double majoring was when I started discovering suddenly what’s going on with DNA and Biotechnology?

Now Biotechnology is a really big field. But what really captured my interest, was this idea of what’s called Genetic Engineering, which is all about taking traits from some other creature and putting them into a new one. It’s bringing the world of corn books even closer to reality.

So I’ll start off with discussing some of what Genetic engineering is all about. And previewing the basic process of Genetic Engineering. I’ll also discuss some of the amazing purposes that Genetic Engineering has been put to. Then we'll dive into the details of how do we make the recombinant DNA, or the combination of DNA from different sources that genetic engineering is all about. Then we'll discuss how do you insert that into whatever a new host is going to be carrying this new set of DNA. And last how the heck do we check to see whether or on you’re successful, something called checking for expression.

So I’d say that genetic engineering is all about taking DNA from one organism and putting it into another. But, how and why would you do that? Well if you're Peter Parker the why is pretty simple. You want supper powers, you want spider powers. But it doesn’t work out that in the real world, or does it?

For diabetics, their daily injections of insulin are what keep them alive. Back in the 1920s scientist figured out how to actually get insulin.

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And what they did is that, they were using the pancreases from dead pigs and cows, and grinding them up to get insulin. The problem was because it’s not human insulin, some people were allergic to it, and would suffer bad symptoms or side effects from it. The other big problem is that,it took about eight thousand pounds of pancreases to get one pound of insulin. So it wound up being both dangerous and really expensive.

In the mid late 70s however some scientist figured out how to get the sequence of DNA in a human, and put that into a bacteria. Now a bacteria don’t sit there and say, “Oh this is human I can’t use it.” No, they follow whatever instructions are in the cell. And so these bacteria just start cranking out the protein they were told to which was human insulin.

Nowadays there is massive, vast chock full of bacteria pumping out gallons and gallons of insulin. It’s easily separated out. And this means that we’ve got this supper hero bacteria, that are saving the lives of millions of diabetics every where, with this human insulin.

What are some of the other uses of genetic engineering? Well in the world of agriculture, genetic engineering has been this major development. One of the most common things done in agriculture, is the creation of corn and other plants that have a genetically engineered protein in them. That produces small levels of pesticide in the actual plant itself. That means here is a normal plant and you can see bugs have been eating it destroying it. While this one, the bugs take a few bites and die.

This has, for those who are really in favor of it, this has revolutionized agriculture, because the farmers don’t have to spray nearly as much pesticides. Those who are against it are kind of worried about what will this do, putting this new gene in the ecosystem do. Organic farmers also are a little bit antsy about this, because it actually turns out that the pesticide that’s being used, is something that’s created by a bacteria. And organic farmers don’t use pesticides sort of.

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What they do is they spray on a bacteria which is natural, and then that bacteria makes the pesticides. And so that’s the one pesticide they’re allowed to use. And they’re worried that the use of it by massive agricultural companies, will wind up making evolution occur and the bugs will stop being stopped by it.

Now, what are some of the other uses in agriculture? Well it ranges. They’ve added some frost resistant genes to crops to make them not be damaged by frost. They’ve added genes from other sources that make the crops last longer or stay ripe longer. They’ve even started doing the stuff where they find the DNA used by spiders, to make spiders silk. And they’ve even inserted that into goats. Now maybe you’re thinking, why do you want? Are you trying to make spider goat? No. Isn't some goat running around shooting webs from its udders, because that will be utterly impossible. Sorry for the chizzy joke, I thought I’d milk that one for all I could.

But it turns out instead, what they’re doing is that, that goat is all about making milk. And in the milk there is a lot of protein. Well with the spider silk protein, the goat makes it and it’s in amongst all the other proteins that are in the milk. And we’ve had centuries and centuries of technology all about separating proteins in search for milk. We call that making cheese and yoghurt. And so this is a great way to get bucket loads of the spider silk protein.

Now why would we want to that? Well, if you’ve read a comic book, you know spider silk is much stronger and lighter than the equivalent amount of steel. And so it’s been proposed to use this to generate things such as very light weight bullet proof vests, using it to make new body forms for cars and aircraft. The possibilities are endless and it’s much better idea than trying to, I don’t know. It’s kind of creepy to imagine hurting spiders, and would you want to milk one?

[0:06:00]
What about medicine? Besides drugs, they’ve been used to do a lot of new techniques. Such as creating this genetically engineered mice here called knock-out mice. Where, a gene has been turned off. Now this is a region where you think, "Wow, there is all these genetic diseases that people have, why don’t we just genetically engineer some people to fix their problems?" Works in concept, in reality it gets pretty complicated because, this process which is called gene therapy, has had some problems in the past. Because we're having problems targeting exactly the genes that we’re trying to change. And a lot of times it’s accidentally caused things to go wrong like cancer.

One of the first examples of it being used successfully, is to deal with a problem called Server Compromised Immunodeficiency problem or SCIDS. Some of you may have heard of this boy in the bubble syndrome. And it’s a problem with one gene in your body. And this happens to be however the gene that turns your immune system on. If you've got a bad version of that gene then your immune system never turns on. And so, that’s why it’s called boy in the bubble disease because one kid who suffered from this, spent his entire life essentially in a bubble.

Well, some scientist figured out how to take out the cells from some of these poor children, who were suffering this terminal disease. The one reason it's considered bio-ethically okay to experiment on them, because they’re going to die anyway. And what they did is that, they took out the cells, and then they used a modified virus.

Now viruses, a lot of them work by sticking their DNA into your cells. And what they did is they took the virus, got rid of the disease causing DNA, and instead stuck in the new gene. To give these kids the working version of the ADH gene, that their bodies couldn’t make. Then, they took the virus, mixed it in with some of their bone marrow cells, allowed those cells plenty of time to grow. And then they inserted the bone marrow cells back into the kids, turns out it’s working.

[0:08:00]
So this is the basic idea. You cut the DNA from a source that you want, a donor. Then you add it to your vector DNA, that’s the virus DNA in this case. And then you put it in to your cells and hope that it works.

Sounds easy and overall it is. But, like with most things, the devil is in the details. So let’s take a closer look at that first step i.e. how do you join the donor and vector DNA together?

Now the whole process of combining DNA together i.e. making recombinant DNA, depends on a pair of proteins or enzymes. Those are restrictions enzymes and DNA ligase. Restriction enzymes are special proteins found in bacteria that are used by the bacteria to chop up viral DNA before the virus has a chance it kill the bacteria.

What’s special about restriction enzymes is they go along looking for very specific sequences called recognition sequences, or restriction sites. And they’re special in that they’re palindromes. If we take a look, this one for example, GAATTC. You’ll notice, reading the other direction, is exactly the same GAATTC. Because you remember, DNA is oriented in anti-parallel directions. And so what happens is that the restriction enzyme will go along and it'll snap the sugar phosphate backbone or break it, between typically the first nucleotide in the sequence, and then the next ones.

What this does is it leaves a little over hanging section of single stranded DNA. Now, these single stranded pieces of DNA could easily pair up back with each other., because of hydrogen bonds. Or they could also do it to any other sequence of DNA, that’s been cut with the exact same restriction enzyme, because they leave identical overlapping ends.

[0:10:00]
And this tendency to stick together, is why they’re called sticky ends. Now you’re maybe wondering, well what’s the point? Well the thing is, if you can this vector DNA, the DNA you can use to get it into this cell that you’re trying to genetically engineer. If you cut your vector DNA with the exact same enzyme as you used to cut the donor DNA; the DNA that you want to take a new gene out of, then you can mix them together.

So let’s take a look at that. Here we see some donor DNA, and we chopped it with our restriction enzyme. Here is our vector DNA. We cut it with the exact same enzyme. So now we mix them together and we’ll see that the sequence here, well hydrogen bond together and there. Now the challenges, yeah they’re hydrogen bonded together, but how do you get them to stay there? Because you guys know hydrogen bonds while they’re numerous there’s lots of them, they’re individually weak. That’s where that DNA ligase enzyme comes in.

DNA ligase is an enzyme used during DNA replication to join Okazaki fragments together. Or it’s also used to repair any accident or damage done to your DNA. Well, after you’ve put together your donor DNA, and your vector DNA, takes you some heat and denature the restriction enzymes. And then, mixing your ligase enzyme. The ligase will go along and any place where there’s some fragments sticking together, it will fix together the sugar phosphate bonds here and here, thus creating one new DNA molecule that’s made of these two different sources i.e. recombinant DNA.

Now previously I had talked about using a virus as your vector DNA. When we talked about how they’ve doing gene therapy. Another common vector DNA is something called a plasmid. If we take a look at those, you can that a plasmid is one of the small circles of DNA that is often found in bacteria.

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Bacteria again, don’t care the DNA that’s inside of him comes from. And because their own chromosome is circular, they leave the plasmids alone. And so what will happen is, if you can get a plasmid with your recombinant DNA into the bacteria, then it will follow the interactions you just gave it. In fact nowadays, you can open up catalogues and you can live through gazillions of pre-made plasmids that come already generated.

What will happen is that, you’ll have a gene here on the pre-made plasmid. That gives the bacteria with that plasmid gives it the ability to survive any particular antibiotic you want. That allows you to then later on select to see which cells got the plasmid verses which ones don’t.

These other things here are various regulatory sites that help you turn on or turn off the genes that you're adding to the bacteria. This is an awesome ability. And you’ll notice also there’s other little sites indicated. These will be locations or restriction enzyme locations that allow you to chop up your plasmid with number of the different commonly available restriction enzyme. So you can make whatever recombinant DNA you want.

So now that we’ve got a recombinant DNA now the challenge is getting it into the cell. If you'd used a virus as your vector, then the process is as simple as just letting the virus infect the cell that you’re trying to get it the DNA into, which is called the host cell. If you’re using a plasmid on the other hand, then you have to do a process called transformation.

Lets take a quick look at that. So here again we’ve got our vector plasmid. We chopped it open using our restriction enzyme, and here is our DNA that we’ve chopped and now we want to get this dark green donor DNA, the desired gene, together with the vector. We add in our ligase, and now we do transformation. Transformation is the process of getting a plasmid into a bacterial cell.

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Now, the general way that you this, one of the most common techniques is a process known Hitchcock. And basically it just involves, you put your test tube of bacteria onto some ice. You let it sit for a while then put it into hot water, and then put it back on ice. Previously, you have to treat the bacteria generally to try to poke holes in its membrane. This called making the bacterial cells competent. This is often done either using electricity or adding something like calcium chloride.

So once you’ve done that, you give the cell a couple of minutes to relax. And then recover, and then you add it to a test tube chock full of food. And what will the bacteria do? Well let’s take a look.

As it takes in food, it does what bacteria do and when they eat. And what people do, eventually. They start to make babies. And every time you go from one bacteria to two bacteria, it faithfully copies all of i’s DNA. Whether is its own chromosome or the plasmid, this will wind up giving you a test tube chock full of clowns of the original modified bacteria.

So no matter if you do transformation or just the viral infection. You can never guarantee a hundred percent effectiveness. And so that’s when you have to view this thing that’s called checking for expression. Now remember we did this transformation with a plasmid that has an antibiotic resistance gene. Well then we just squatted that bacteria that we allowed to grow up, we squat them onto a Petri dish.

Now in this Petri dish we added in a bunch of the antibiotic, that the plasmid carried the resistance gene too. And this allows us to kill off any of the bacteria that did not absorb the plasmid that we’re trying to get into them. So of the thousands and millions of bacteria that are squatted on here, you can see that there is only maybe ten or so survivors. Now, do all of them have the inserted donor DNA that we like? No. But randomly some of them should.

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So what you can do is you can start looking to see, what are these different cells doing. And there is a number of ways you can do that. One is that you put some filter paper on top. That bits of the product being produced by this bacteria soak into the filter, and then examine that to see which one's got it. But hopefully something that you can do, is that you can turn on those genes. Again remember, that you’re working with pre-made plasmids that have all this genetic things, genetic regulatory sections. And a lot of times you’re adding on a gene that turns the color. And so you can just go the green ones got it. And that's it. What you’re doing is you’re checking to see whether or not it worked.

So I’ve glossed over some of the details, things like how bacteria don’t have introns and exons like we eukaryote do. So, in reality you need to spend some time editing out the introns, creating just a series of DNA that has the exons. This is called cDNA which is short for complementary DNA. But in general, as you’ve seen, it's not that hard of a process. Let’s review by taking a quick look at this really nice YouTube video.

And we’ll go ahead and we'll make it nice and big so we can see it on the screen. So we'll go ahead and we’ll start it up. What we see here is a test tube chock full of the plasmids or vector DNA. When we zoom in, we’ll see in this test tube is gazillions of these plasmids floating around. Every plasmid will have the antibiotic resistance gene, something called the replication origins or actually will get copied. And then this is the area where we can cut in and paste in our donor DNA. So now we’re going to go ahead and bring in our restriction enzymes, so we can start chopping up the DNA. And here we see a recognition sequence. So we use our restriction enzymes and they chop it up. Remember, those sticky ends ,use that during an AP Biology essay and you got yourself a point.

[0:18:00]
Here comes in our donor DNA. Now instead of having one copy or thousands of copies of the desired sequence, instead we'll have single copies of thousands of different regions of DNA, because we’re not sure where that gene is. But we mix them together, they've both been cut with the exact same enzymes. The hydrogen bonds start to form now we need to add in the ligase enzyme. And now we’ve got our donor DNA added to our vector DNA. This is now called recombinant DNA.

Now if you remember, what do we have to do next? Put it into the bacteria or transformation. Here you can see a sample of other plasmids each with their own different kind of donor DNA. Here is our bacteria our E. coli. So we go through that Hitchcock process to get the DNA into them. And you’ll see, some of the bacteria will take it in, others won't. How it goes in is kind of random. This guy, the plasmid wounded up inside. This one here, these guys not at all. And what that means is that these guys with the recombinant plasmids in them, are antibiotic resistance.

So here we have our test tube and we’re going to go ahead and we’re going squat that out into our Petri dish. Remember, far less that 10% of them actually got our plasmid. So there will be a massive horrible death and destruction of bacteria on our Petri dish, but we don’t care. They’re bacteria. We pour it on and bacteria start dying. We incubate it at 37 degrees Celsius that’s human body temperature, which is the ideal environment for E. coli that live in humans.

So these guys have a plasmids this guys don’t. So what’s going to happen? They’re going to die. These guys that did get our plasmid, they'll start eating, especially now that we’ve removed their competition. And once they start doing that, they’ll start making more and more babies.

[0:20:00]
As you can see, every time they get ready for the cell division, they copy off the plasmid. Making lots of copies of the plasmid, enough for each daughter cell. And we can see that process we’re going to undergo binary fission, which is cell division for bacteria. So we’ve gone from to two bacteria, and we just keep doing that over and over. Each bacteria is taking in nutrients, it's following the instructions of its chromosome and its plasmid.

And so on the surface of the Petri dish, you’ll start to see more and more colonies growing. Initially they’ll start of microscopic, but as you wide up making millions of copies of each bacteria, and these clusters or colonies or sometimes are called clones, they’re all genetically identical. And each of them will be producing whatever gene we inserted into it.

So that’s it, that’s the process. I highly recommend you go through this a couple of times until you get it. Because it is very likely to appear on the AP Biology exam. Whether in the multiple choice or in the essays. But once you’ve mastered it, you’ve mastered one of their favorite essay questions. So good luck.