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New DNA (gene)
Plasmid DNA
Restriction
Enzymes “cut”
Plasmid DNA
Piece of DNA is
Removed
New Piece (gene)
of DNA is
“stitched” to
Plasmid DNA
Bacterial Proteins that Cut Both Strands of the DNA Moloecules
Small Ring of DNA Found in a Bacteria Cell
E. Coli cell
Plasmid DNA
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 by
inserting a gene that will make the bacteria
resistant to the antibiotic, ampicillin
Ampicillin:
A chemical that kills bacteria
Plasmid:
A circular piece of DNA found in bacteria
pGREEN:
A plasmid that contains genes that protects bacteria
from ampicillin and makes the bacteria turn grenn
LB broth:
Food for bacteria
LB plate:
An Agar plate to grow bacteria on
LB/Amp plate: An Agar plate that contains ampicillin
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 with gene
normal growing conditions
bacteria without gene
antibiotics applied
bacteria without gene
normal growing conditions
- pVIB
LB
+ pVIB
LB
bacteria without gene
normal growing conditions
bacteria with gene
normal growing conditions
+ pVIB
LB/ Amp
- pVIB
LB/ Amp
bacteria without gene
antibiotics applied
bacteria with gene
antibiotics applied
Data Table: Bacterial Growth 72 hours after inserting the pVIB
plasmid (Mr. Duane’s classes)
LB / - pVIB
LB / + pVIB
3
LB &Amp /
- pVIB
1
LB & AMP /
+ pVIB
2
Brooks
& Tom
Sid &
Ben
Steve &
Chris
Trevor
James
Beau
3
3
3
1
2
3
3
1
2
3
3
1
2
3
3
2
2
Taylor
& Matt
Bobby
& Alex
Brian &
Casey
Chas &
Zack
Scot &
Jesse
Jimmy
3
3
1
2
3
3
1
3
3
1
1
1
3
3
1
1
3
3
1
3
3
3
2
2
Lars &
Willie
3
3
1
2
Key: 1 – indicates no bacterial growth
2 – indicates some bacterial growth
3 – 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?