KEY CONCEPTS: Genetic transformation, plasmid DNA, cloning ...
Plasmid DNA Restriction Enzymes “cut” Plasmid DNA Piece of DNA is Removed New Piece (gene) of...
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Transcript of Plasmid DNA Restriction Enzymes “cut” Plasmid DNA Piece of DNA is Removed New Piece (gene) of...
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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)
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Bacterial Proteins that Cut Both Strands of the DNA Moloecules
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Small Ring of DNA Found in a Bacteria Cell
Plasmid DNA
E. Coli cell
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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
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To transform the DNA of E. Coli bacteria byinserting a gene that will make the bacteria resistant to the antibiotic, ampicillin
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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
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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.
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1. Practicing sterilizing technique
2. New tip for micropipette
3. Preparing calcium chloride solution
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4. Sterilizing inoculating stick
5. Scraping E. Coli bacteria
6. Adding E. Coli to CaCl2 solution
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7. Inserting pVIB (plasmid DNA) into E. Coli
8. Inoculating Agar plates with genetically transformed E.Coli
9. Spreading bacteria evenly on Agar plates
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bacteria with gene antibiotics applied
bacteria without gene antibiotics applied
bacteria without gene normal growing conditions
bacteria with gene normal growing conditions
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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
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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
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•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?