1. Unit 4 - Code of Life Part I: Genetic Engineering
Biotechnology
Part I: Genetic Engineering

2. Unit 4 - Code of Life
Recombinant DNA
Purpose: To insert genes of one organism into another organism

3. Recombinant DNA Applications
Unit 4 - Code of Life
Recombinant DNA Applications
Green-glowing aquarium fish (jellyfish genes)

4. Recombinant DNA Applications
Fast-growing fish (Salmon with Pout genes)

5. Recombinant DNA Applications
Herbicide-resistant crops (Round-Up Ready)
Molecular basis for the herbicide resistance of Roundup Ready crops.Todd Funke�, Huijong Han�, Martha L. Healy-Fried�, Markus Fischer�, and Ernst Schbrunn�,ｧ+Author Affiliations.�Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045; and.�Department of Organic Chemistry and Biochemistry, Technical University Munich, D Garching, Germany.Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved July 12, 2006 (received for review May 3, 2006) Next SectionAbstractThe engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate has greatly improved agricultural efficiency worldwide. Glyphosate-based herbicides, such as Roundup, target the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely required for the survival of plants. Roundup Ready plants carry the gene coding for a glyphosate-insensitive form of this enzyme, obtained from Agrobacterium sp. strain CP4. Once incorporated into the plant genome, the gene product, CP4 EPSP synthase, confers crop resistance to glyphosate. Although widely used, the molecular basis for this glyphosate-resistance has remained obscure. We generated a synthetic gene coding for CP4 EPSP synthase and characterized the enzyme using kinetics and crystallography. The CP4 enzyme has unexpected kinetic and structural properties that render it unique among the known EPSP synthases. Glyphosate binds to the CP4 EPSP synthase in a condensed, noninhibitory conformation. Glyphosate sensitivity can be restored through a single-site mutation in the active site (Ala-100萌ly), allowing glyphosate to bind in its extended, inhibitory conformation.

6. Recombinant DNA Applications
Unit 4 - Code of Life
Recombinant DNA Applications
Pest-resistant crops (Bt toxin)

7. Recombinant DNA Applications
Unit 4 - Code of Life
Medicine production (bacteria can make insulin, hGH, etc)

8. Making Recombinant Bacteria
Important players:
Gene of interest (i.e. insulin, hGH, etc)
Restriction enzymes – cut the DNA
Plasmid – circular DNA found in bacteria
Bacteria

9. What are Restriction Enzymes?
Target very specific base sequences
Are found in more than 100 different varieties
Are used in nature to protect bacteria from foreign invaders

10. What are Restriction Enzymes?
Unit 4 - Code of Life
What are Restriction Enzymes?
Each restriction enzyme recognizes a very specific nucleotide sequence
EcoR1 recognizes: GAATTC CTTAAG
The enzyme cuts it: G AATTC CTTAA G

12. Unit 4 - Code of Life

13. Making Recombinant DNA
Unit 4 - Code of Life
Making Recombinant DNA
Plasmids - self-replicating rings of DNA containing 2-30 genes, found in bacterial cells

14. Making Recombinant DNA
Unit 4 - Code of Life
Making Recombinant DNA
Isolate the gene of interest using a restriction enzyme
Cut the plasmid (using the same enzyme)
Insert gene into the plasmid
Insert the plasmid into bacteria
Grow bacteria
Harvest & purify the protein

15. Unit 4 - Code of Life
1. Isolating the gene
Use restriction enzymes to cut the DNA strand at specific places that surround the gene

16. 2. Cut the plasmid
Cut the plasmid using the same restriction enzyme

17. 3. Put the gene in the plasmid
Since the gene and the plasmid have the same sticky ends, the gene will stick to the plasmid

18. Unit 4 - Code of Life
The gene of interest is attached to a promoter and some kind of marker

19. 5/6. Put plasmid in bacteria
Insert plasmid into bacteria
Let bacteria make the protein. Harvest protein & purifty it.

20. Unit 4 - Code of Life Part II: DNA Fingerprinting
Biotechnology
Part II: DNA Fingerprinting

21. Unit 4 - Code of Life
DNA Fingerprinting
Definition: Method of visualizing the differences in DNA samples
Purpose:
Forensic science
Determining paternity
Research
Diagnosing Disease

22. Unit 4 - Code of Life
Gel Electrophoresis
Separates DNA fragments by size using electric current

23. Gel Electrophoresis Larger fragments move more slowly
Unit 4 - Code of Life
Gel Electrophoresis
Larger fragments move more slowly
Bands of fragments result

24. DNA Fingerprinting Method
Unit 4 - Code of Life
DNA Fingerprinting Method
Use restriction enzymes to cut the DNA
Unique DNA sequences produce unique banding patterns on the gel
Load the DNA onto agarose gel for gel electrophoresis
Analyze the banding pattern

25. Unit 4 - Code of Life

26. Gel Electrophoresis – Paternity Test

27. Gel Electrophoresis – Crime investigation

28. RFLP Analysis RFLP – Restriction Fragment Length Polymorphism:
Restriction Enzymes used on two (or more) samples of DNA
Different DNA fragments are produced because of variation in the DNA sequences
Genetic markers are used for fingerprinting
Gene sequences are used for genetic tests

29. A single nucleotide change can make a difference
Wild-type allele
AGATCT
TCTAGA
Restriction site
Mutant allele
AGAGCT
TCTCGA
Not a restriction site

30. Unit 4 - Code of Life
– NOTE - WARN STUDENTS OF THE END WHERE THE AFRICAN-AMERICAN BOY IS MAKING FRIED CHICKEN (MY AASP STUDENTS THOUGHT THIS WAS RACIST)