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

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

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

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

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

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…

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

Bacterial Transformation Procedure

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

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.

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