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Biology: Use Restriction Enzyme to Create a Vector

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About this Lesson

  • Type: Video Tutorial
  • Length: 9:25
  • Media: Video/mp4
  • Use: Watch Online & Download
  • Access Period: Unrestricted
  • Download: MP4 (iPod compatible)
  • Size: 102 MB
  • Posted: 07/01/2009

This lesson is part of the following series:

Biology Course (390 lessons, $198.00)
Biology: Biotechnology (16 lessons, $23.76)
Biology: Plasmids and Gene Cloning (4 lessons, $6.93)

Taught by Professor George Wolfe, this lesson was selected from a broader, comprehensive course, Biology. This course and others are available from Thinkwell, Inc. The full course can be found at http://www.thinkwell.com/student/product/biology. The full course covers evolution, ecology, inorganic and organic chemistry, cell biology, respiration, molecular genetics, photosynthesis, biotechnology, cell reproduction, Mendelian genetics and mutation, population genetics and mutation, animal systems and homeostasis, evolution of life on earth, and plant systems and homeostasis.

George Wolfe brings 30+ years of teaching and curriculum writing experience to Thinkwell Biology. His teaching career started in Zaire, Africa where he taught Biology, Chemistry, Political Economics, and Physical Education in the Peace Corps. Since then, he's taught in the Western NY region, spending the last 20 years in the Rochester City School District where he is the Director of the Loudoun Academy of Science. Besides his teaching career, Mr. Wolfe has also been an Emmy-winning television host, fielding live questions for the PBS/WXXI production of Homework Hotline as well as writing and performing in "Football Physics" segments for the Buffalo Bills and the Discover Channel. His contributions to education have been extensive, serving on multiple advisory boards including the Cornell Institute of Physics Teachers, the Cornell Institute of Biology Teachers and the Harvard-Smithsonian Center for Astrophysics SportSmarts curriculum project. He has authored several publications including "The Nasonia Project", a lab series built around the genetics and behaviors of a parasitic wasp. He has received numerous awards throughout his teaching career including the NSTA Presidential Excellence Award, The National Association of Biology Teachers Outstanding Biology Teacher Award for New York State, The Shell Award for Outstanding Science Educator, and was recently inducted in the National Teaching Hall of Fame.

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Founded in 1997, Thinkwell has succeeded in creating "next-generation" textbooks that help students learn and teachers teach. Capitalizing on the power of new technology, Thinkwell products prepare students more effectively for their coursework than any printed textbook can. Thinkwell has assembled a group of talented industry professionals who have shaped the company into the leading provider of technology-based textbooks. For more information about Thinkwell, please visit www.thinkwell.com or visit Thinkwell's Video Lesson Store at http://thinkwell.mindbites.com/.

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Recent Reviews

Nopic_grn
Problem
09/08/2011
~ Azi85

Hi!This video is incomplete. It is 6 minutes instead of 9!

Nopic_grn
Problem
09/08/2011
~ Azi85

Hi!This video is incomplete. It is 6 minutes instead of 9!

One of the questions we want to address is - if I have a plasmid - remember plasmid its that extra chromosomal piece of DNA that you find inside a prokaryotic cell. If I have a plasmid, can I somehow get these out of bacterial cells? And the answer is yes; it's very simple. You simply break open the bacterial cell. And then once you break open the bacterial cell all you have to do is centrifuge them and the plasmids will separate at different levels from the bacteria. Think about it. You've got the plasmid being smaller will separate at a different level than the very large bacterial genomic DNA. So, it's easy to get plasmids out.
So, do we therefore; have a tool now to say could I take these plasmids - maybe suck them up in an eyedropper of some kind and then, like squirt them into another cell. And in essence, do what bacteria do naturally, and put this plasmid from one cell into another. Well, that would be easy. All you would have to do is treat the target bacteria with some kind of chemical to make their cell walls a little porous, and the plasmid would get in there.
Well, what good does that do to you? So, you have plasmids around. So, what. But if I could somehow put something into this plasmid, a gene of choice, now I would have a vector to move my gene. But how are you going to break open a plasmid, and insert something into it?
Well, this is a good question, but it was answered in the 1960s. And that came with the discovery of a group of enzymes made by bacteria actually. And these enzymes are called "restriction enzymes" or officially "restriction endonucleases." You see bacteria are under constant attack. I know you know this that bacteria can be attacked by a creature called the bacteriophage. And that bacteriophage generally will always inject a nucleic acid, generally DNA. And when that DNA goes into the bacteria it will insert into the bacterial genome, and that will therefore, trans-literally make this bacterium make bacteriophage DNA. Remember the generic name for bacteriophage is a virus. Well, bacteria are now slouches. If this was the reality of their life and that's all they had to deal with bacteria would be extinct because bacteriophage would just like kill them. But, it turns out that bacteria have a defense against bacteriophages. And when a bacteriophage lands on a bacteria, some of them make these restriction enzymes - these restriction endonucleases. And what these are - these restriction endonucleases, which I'm going to refer to as RE's cut DNA. So, therefore, the word Endo - inside the nuclease. So, it's a nuclease. Why the word restriction? And now we come to the use of these things in biotechnology.
You see these are not just plain old nucleases. Remember that a nuclease just digests by hydrolysis - DNA or RNA. These are restricted nuclease digesters. And they can only recognize a certain sequence on the DNA. And it's usually about six nucleotides. So, for example, there are hundreds, if not thousands of these restriction endonucleases that you can buy - just order them. And one I want to mention is called EcoR1[. ]Now, first of all, let me tell you how we name restriction enzymes. We name them by the bacteria they were discovered in so this particular one was discovered in E. coli - strain R1. So, it's called EcoR1. I want to just tell you about EcoR1 as my example. EcoR1 is restricted, and it's restricted to a site GAATTC. And obviously that's part of the DNA strand - CTTAAG. And here's the thing. When EcoR1cuts this it will only cut that sequence and here's the second thing I want you to understand. It cuts it in what we call a staggered cut. That's going to loom very importantly in a few minutes. So, what's going to happen is when EcoR1[ ]cuts, now some of them don't do staggered cuts, but this one does, and this is an important one. So when EcoR1[ ]cuts this here's what you are going to get. This strand of the DNA 5335 this strand of the DNA is going to cut like this. So, were going to get G and then we're going to get C, and then that's going to overhang TTAA, and this side is going to look like this so we're going to get AATTCG. We call these sticky ends. In other words, they can go back together again. Now, it will be a little bit weak it would be nice if we had, like remember ligates that enzyme that ligates DNA - puts it together, but we don't have that right now. But, you know, if left to its own devices, this would go back into here.
So, bacteria use these and what they do is they chop up the DNA of the virus so the bacteriophages coming in there, and then there are other nucleases eat them up and they are protected.
What I want to talk to you about is how we can use these enzymes once we isolate them. How cool is this? Imagine if you will that you have a plasmid, and you want to cut that plasmid open. Now plasmids aren't all that big. And, we'll talk more about the odds of getting that to happen later on. But let's just say you can cut this plasmid open with EcoR1[. ]Now when you have that cut open what do you have? You have sticky ends, don't you? Right, because you used EcoR1[. ]Now, let's just say I happen to have, and all the details of this will come overview first. Let's just say I want that gene - that gene right there. And coincidentally that gene is flanked by the EcoR1[ ]recognition site. So there's an EcoR1[ ]recognition site upstream of the gene, and there's an EcoR1[ ]site down stream of the gene.
Well, how cool would it be if I could cut that gene out, and then, since it has this - what enzyme would I use? Well, I cut this one with EcoR1[ ]- let me cut this with EcoR1[ ]because what's going to happen? The sticky ends from this one are going to be the same sticky ends as this. So now, when we mix this with this plasmid look what we can do. Automatically, and throw in some ligates, we're going to be solid - we have just added the gene of choice. Right there we've added this gene and it's sites to this plasmid. We just made a vector. Now imagine that this gene happens to be a gene for antibiotic resistance. Guess what? You just made a plasmid that will give any bacteria antibiotic resistance. Or, let's just say it already have antibiotic resistance, and this happened to be a gene for insulin. Could we really put the gene for insulin into a bacterial plasmid? And could we really take that plasmid and actually put it in a bacterium. And could we get that bacterium to make human insulin? Not only is that a good thought it's already been done.
Biotechnology
Plasmids and Gene Cloning
Using a Restriction Enzyme to Create a Vector Page [2 of 2]

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