Biotechnology Part II. Genetic Engineering and Recombinant DNA.
Biotechnology Recombinant DNA and its Applications.
-
Upload
stephen-chandler -
Category
Documents
-
view
234 -
download
4
Transcript of Biotechnology Recombinant DNA and its Applications.
![Page 1: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/1.jpg)
BiotechnologyRecombinant DNA and its
Applications
![Page 2: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/2.jpg)
Introduction
1) What are Plasmids?
2) How can we modify plasmids? Restriction Enzymes
3) Origins of restriction enzymes.
4) A close look at restriction enzymes.
5) Understanding plasmid diagrams.
![Page 3: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/3.jpg)
What are Plasmids?
• Circular DNA that is used by bacteria to store their genetic information.
• Modifying plasmids to include extra genes allows for the production of new proteins.
In this Lecture…
![Page 4: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/4.jpg)
How Can We Modify Plasmids?
1) Restriction Enzymes BamHI, HindIII,
etc. Where do they
come from? How do they
work? Different
restriction enzymes do different things.
2) DNA Ligase
Restriction Enzyme attached to DNA before cleavage
In this Lecture…
![Page 5: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/5.jpg)
Origins of Restriction Enzymes
1) Bacteria produce restriction enzymes to protect against invading viral DNA/RNA.
![Page 6: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/6.jpg)
Origins of Restriction Enzymes
2) The enzymes cut the invading DNA/RNA, rendering it harmless.
![Page 7: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/7.jpg)
Restriction Enzyme in Action
1) DNA strand with EcoRI restriction site highlighted.
2) EcoRI restriction enzyme added (outline of separation about to occur).
3) Restriction fragments separate, with “sticky ends” at each edge.
Sticky Ends
![Page 8: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/8.jpg)
Adding DNA Ligase
DNA ligase bonds sticky ends cut with the same restriction enzyme. Sticky ends cut with different restriction enzymes will not bond together.
Why? Because the base pair sequence of the two sticky ends will be
different and not match up.
Sticky Ends
![Page 9: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/9.jpg)
Plasmids Can Be Drawn to Show the Genes They Carry
In this diagram: • Blue and Orange
are drawn as genes.
• Triangles are indicating the known restriction sites for a restriction enzyme. (shapes can vary)
• Plasmid Maps are more complex.
Plasmid Name
Bp size
![Page 10: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/10.jpg)
Plasmid Maps Indicate Restriction Sites and Genes
![Page 11: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/11.jpg)
Make Recombinant DNA Using Restriction Enzymes
Application Exercise
![Page 12: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/12.jpg)
DNA From Two Sources(Restriction Sites Labeled)
Circular DNA Linear DNA
![Page 13: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/13.jpg)
Application of Restriction Enzymes
![Page 14: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/14.jpg)
Adding DNA Ligase
![Page 15: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/15.jpg)
Recombinant DNA Plasmid
• Many possible recombinant DNA plasmids can be produced, but this was the desired plasmid for the experiment.
![Page 16: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/16.jpg)
Many Other Recombinant Possibilities
…and many more!
![Page 17: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/17.jpg)
Plasmid DNA Insertion
DNA plasmids can be inserted into bacteria using a variety of laboratory processes.
![Page 18: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/18.jpg)
Transgenic Colony Allowed to Grow
![Page 19: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/19.jpg)
How Do We Get the Desired Plasmid?• Restriction fragments will
ligate randomly, producing many plasmid forms.
• Bacterial insertion would be necessary, then colony growth, and further testing to isolate bacteria with the desired plasmid.
Transformation of bacterial cells through electroporation.
Bacteria are then moved to a growth plate, and grown on selective media to “weed out” cells that have not picked up the desired plasmid.
Recombinant plasmids
![Page 20: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/20.jpg)
Running Digested DNA Through Gel Electrophoresis
Lab Experiment (Part 1)
![Page 21: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/21.jpg)
Goals of this Hands-On Lab• Take plasmid DNA that has
been previously cut with restriction enzymes and compare that to a plasmid NOT cut with restriction enzymes, by running them through a gel.
• Look for different banding patterns and understand how to read them.
• Predict what kind of banding pattern a plasmid will make based on: 1. The restriction enzyme used.
2. The plasmid’s structural shape.
![Page 22: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/22.jpg)
Gel Box Loading Techniques
• Look directly down the axis of the pipette.
• Loading dye makes the sample heavy, but it can still easily swish out of the well.
• Squirt down slowly.• Take the tip out of the buffer.• Then release the plunger.• If you don’t do that, you will
suck the sample back up.
![Page 23: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/23.jpg)
Add DNA samples and ladder to the wells and “run to red!”
![Page 24: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/24.jpg)
10 kb
8 kb
6 kb
5 kb
4 kb
3 kb
2 kb
1 kb
.5 kb
Sample fragments move toward positive end.
![Page 25: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/25.jpg)
Analyzing Your Gel
Lab Experiment (Part 2)
![Page 26: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/26.jpg)
What Makes Up the Banding Pattern in Restricted DNA?
• The restriction enzyme cleaves the DNA into fragments of various sizes.
• Each different size fragment will produce a different band in the gel.
• Remember that fragments separate into bands based on size.
Lancer Plasmid
6700 Bp
3300 Bp
2000 Bp
1400 Bp
![Page 27: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/27.jpg)
What Makes Up the Banding Pattern After Adding DNA Ligase?
• Several combinations of plasmids will result from the reaction.
• The many forms will contribute to different bands.
(See following slides for chemical and structural forms)
![Page 28: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/28.jpg)
Different Recombinant Forms
• Adding DNA Ligase does not always make the desired plasmid!
• Few if any could be what you wanted.• Think about the large number of possible
combinations.
![Page 29: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/29.jpg)
Different Structural Forms
circle
“nicked-circle”
“multimer”
Different structural forms produce different bands.
Nicked Circle
SupercoiledLinear
![Page 30: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/30.jpg)
What Are Some Applications of Recombinant DNA Technology?
Bacteria, Yeasts, and Plants can all be modified to produce important pharmaceuticals, enriched foods, and industrial products.
![Page 31: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/31.jpg)
Assignment
• Find a product in any of the fields listed in the previous slide and write a short summary to include:– The organism used– The recombinant DNA product– The product’s application– Is the product an improvement compared to
similar non-recombinant products (explain)?
![Page 32: Biotechnology Recombinant DNA and its Applications.](https://reader036.fdocuments.in/reader036/viewer/2022062308/56649d8a5503460f94a703fa/html5/thumbnails/32.jpg)
10 K
b L
add
er
5 Kb
Multimer
NickedSuper Coiled
Linear Fragment
A- A+
Linear Fragment
10 K
b L
add
er
10 K
b L
add
er