1. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid

2. What is a plasmid? A small circular piece of DNA Naturally occurring
Can be altered in lab to express protein of interest

3. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid

4. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid

5. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Bacterial chromosome

6. What is Transformation?
Bacterial chromosome
Uptake of foreign DNA, often a circular plasmid
Plasmid
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Bacterial chromosome
Bacteria now express cloned protein…

7. How are plasmids engineered?
DNA Plasmid Vector
Host DNA fragments
(i.e. coral or jellyfish FP coding DNA)
Ligate (paste) fragments into cut DNA vector
Cut plasmids open with restriction enzymes
Cut genomic DNA into fragments +
Screen for and select plasmid containing gene of interest

8. Bacterial Transformation Procedure

9. Shielding the charge CaCl2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++ O
CH2 P
Base OH
Sugar

10. Stress through heat Incubate on ice slows fluid cell membrane
Heat-shock increases permeability of membranes
Leave in heat 45 seconds!!!
Too short, and bacteria won't let in plasmid.
Too long, and the bacteria will die.

11. Why Antibiotics?
Antibiotics inhibits cell growth. Only cells that can inactivate the Antibiotics around them will grow.
Antibiotics is a selection mechanism that only allows transformed bacteria to grow on the plate