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)

Bacterial Proteins that Cut Both Strands of the DNA Moloecules

Small Ring of DNA Found in a Bacteria Cell

Plasmid DNA

E. Coli cell

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

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

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

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 / CaCl2 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.

1. Practicing sterilizing technique

2. New tip for micropipette

3. Preparing calcium chloride solution

4. Sterilizing inoculating stick

5. Scraping E. Coli bacteria

6. Adding E. Coli to CaCl2 solution

7. Inserting pVIB (plasmid DNA) into E. Coli

8. Inoculating Agar plates with genetically transformed E.Coli

9. Spreading bacteria evenly on Agar plates

bacteria with gene antibiotics applied

bacteria without gene antibiotics applied

bacteria without gene normal growing conditions

bacteria with gene normal growing conditions

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

- pVIBLB/ Amp

+ pVIB LB

- pVIBLB

Data Table: Bacterial Growth 72 hours after inserting the pVIBplasmid (Mr. Duane’s classes)

LB / - pVIB LB / + pVIB LB &Amp /- pVIB

LB & AMP /+ pVIB

Brooks& Tom

3 3 1 2Sid &Ben

3 3 1 2Steve &Chris

3 3 1 2TrevorJames

3 3 1 2Beau 3 3 2 2Taylor& Matt

3 3 1 2Bobby& Alex

3 3 1 3Brian &Casey

3 1 1 1Chas &Zack

3 3 1 1Scot &Jesse

3 3 1 3Jimmy 3 3 2 2Lars &Willie

3 3 1 2

Key: 1 – indicates no bacterial growth2 – indicates some bacterial growth3 – indicates a lot of bacterial growth

•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?