1. Chapter 9: Biotechnology and Recombinant DNA

2. BIOTECHNOLOGY
Use of microorganisms, cells or cell components to make a product
Now: being used as "factories" to make chemicals not naturally made in the cells
Inserting genes......recombinant DNA (rDNA) technology
Genetic Engineering

3. rDNA Technology
Gene Cloning
Restriction Enzymes

4. Insulin-the First Recombinant Protein
Nobel Prizes for insulin:
discovery of insulin; patent for insulin sold for one dollar
the first protein with the determined amino acid sequence
the first protein with determined spatial conformation (crystallography)
Recombinant insulin
1978- human insulin produced in E. coli

5. How to produce human protein in bacteria?
What do you need to make a protein?

6. Gene Cloning The goal: amplify a given DNA sequence in bacterial cells
Steps:
Gene isolation
Isolation of vector
Manipulation of DNA sequence
Cutting -Res
Splicing - DNA ligase
Transformation of bacteria
Selection of “correct” bacteria

7. Restriction Endonucleases
Enzymes that cleave DNA at a specific sequence (usually 4, 6 8bp palindromes)
Some leave “sticky” ends, some leave “blunt” ends
More than 800 are currently available
EcoR1

8. How do you get the protein out?
E. coli and some other bacteria
Cells must be broken open and the product purified
expensive
Some bacteria secrete the product
Can be removed from the medium
Yeast-able to continuously secrete product
“eukaryotic workhorse of biotechnology”

9. Other tools of biotechnology
PCR
Gel Electrophoresis

10. Polymerase Chain Reaction
PCR: small samples of DNA are amplified quickly
Can make billions of copies in a few hours
Thermalcycler: set for the desired temp, times, and number of cycles
Uses DNA pol from thermophilic bacterium
Thermus aquaticus
Can survive the intense heat

11. Gel Electrophoresis Used to separate DNA using an electric field
Separate based on size