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?