1. Recombinant DNA

2. Restriction Enzymes
Enzymes that break down DNA by cutting it at a specific site (restriction site)
Originally used as cellular defense against invaders
Used in Biotech to create recombinant DNA or for DNA fingerprinting
Each enzyme has a unique restriction site that it recognizes

3. Example of Restriction Sites
EcoRI
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATC 3’
3’ CTTA G 5’
“Sticky ends” (sections of single stranded DNA) are created.

4. Time to Practice
DNA Scissors Activity will enable you to visualize how restriction enzymes work!

5. What is Recombinant DNA?
DNA that contains a gene or genes from more than one organism

6. What are some uses for recombinant DNA?
Create GMO’s
Protein production
Organisms with specific traits

7. How do you create recombinant DNA?
Identify desired gene and cut it out with restriction enzymes. (sticky ends)
Cut a plasmid with same restriction enzyme.

8. Join your gene to plasmid and seal with ligase to form a recombinant plasmid

9. Put recombinant DNA into a host cell to replicate.
Detect cells that have recombinant DNA and can make the desired product.
Example – antibiotic resistance or ability to glow

10. How does this relate to class?
Using recombinant plasmid pGLO in transformation lab
Contains Green Fluorescent Protein gene (GFP) that glows green in UV light and is ampicillin resistant

11. Why is this important in the real world? (not just in science class)
Real uses of recombinant DNA?
Ethical issues?

12. Time to Practice!
We are going to practice creating recombinant DNA using paper plasmids. If you master this, you will get to make real recombinant plasmids during our transformation lab.

13. Transformation Lab Vocab
Sterile Technique
Proper way of handling bacteria to avoid contamination.
Streaking
Process of spreading bacteria on an agar plate to separate the cells

14. Agar Arabinose Ampicillin
Substance on plate that provides support and food for bacterial growth
Arabinose
Added to Agar plates, sugar that’s food for bacteria
Ampicillin
Added to Agar plates, Antibiotic that kills bacteria

15. Bacterial Library Colony Cloning
A collection of bacterial cells that contain recombinant plasmids with random DNA inserts from a single species
Colony
A clump of genetically identical cells growing on an agar plate (clones)
Cloning
When a population of cells is prepared by growth from a single cell (entire population is genetically identical)