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Transcript
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?