2. Plasmid DNA Restriction Enzymes “cut” Plasmid DNA Piece of DNA is Removed New Piece (gene) of DNA is “stitched” to Plasmid DNA New DNA (gene)

3. Bacterial Proteins that Cut Both Strands of the DNA Moloecules

4. Small Ring of DNA Found in a Bacteria Cell Plasmid DNA E. Coli cell

5. Bacteria Breaks Down Pollutants into Harmless Products Bacteria Extracts Minerals from Ores Insert the Human Gene into Bacteria to Produce Insulin for Diabetics Produce Artificial Sweeteners

7. To transform the DNA of E. Coli bacteria by inserting a gene that will make the bacteria resistant to the antibiotic, ampicillin

8. Ampicillin:A chemical that kills bacteria pGREEN:A plasmid that contains genes that protects bacteria from ampicillin and makes the bacteria turn grenn Plasmid: A circular piece of DNA found in bacteria LB plate: An Agar plate to grow bacteria on LB/Amp plate: An Agar plate that contains ampicillin LB broth: Food for bacteria

9. 1. Use micro-pipets and innoculating sticks to mix calcium chloride solution with E. Coli bacteria. 2. Label 4 Agar plates. LB +pVIB LB – pVIB LB/Amp + pVIB LB / Amp - pVIB 3. Mix pVIB plasmid with appropiate bacteria / CaCl 2 solution. 4. “Heat shock” bacteria in hot water bath and ice so that it takes in plasmid. 5. Spread + pVIB bacteria on “+” Agar plates 6. Spread – pVIB bacteria on “-” Agar plates 7. Incubate at room temperature for 72 hours and record bacterial growth.

10. 1. Practicing sterilizing technique 2. New tip for micropipette 3. Preparing calcium chloride solution

11. 4. Sterilizing inoculating stick 5. Scraping E. Coli bacteria 6. Adding E. Coli to CaCl 2 solution

12. 7. Inserting pVIB (plasmid DNA) into E. Coli 8. Inoculating Agar plates with genetically transformed E.Coli 9. Spreading bacteria evenly on Agar plates

13. bacteria with gene antibiotics applied bacteria without gene antibiotics applied bacteria without gene normal growing conditions bacteria with gene normal growing conditions

14. bacteria with gene antibiotics applied bacteria without gene antibiotics applied bacteria with gene normal growing conditions bacteria without gene normal growing conditions + pVIB LB/ Amp - pVIB LB/ Amp + pVIB LB - pVIB LB

16. What are the controls for this experiment? Why is it important to practice sterilizing technique? Did the students successfully insert the pVIB gene? Why would you sometimes take antibiotics?