Bacterial Transformation
What is transformation? Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance
What is a plasmid? A circular piece of autonomously replicating DNA ori bla Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest
pGlo plasmid Beta Lactamase Green Fluorescent Protein Ampicillin resistance The beta lactamase protein is produced and secreted by bacteria that contain the plasmid. Inactivates ampicillin. Green Fluorescent Protein Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription pGLO ori bla GFP araC
Bacterial Transformation GFP Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial chromosomal DNA Cell wall
Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat Shock Chemically-competent cells uptake DNA after heat shock
Transformation procedure Suspend bacterial colonies in Transformation Solution Add pGLO plasmid DNA Place tubes on ice Heat shock at 42oC and place on ice Incubate with nutrient broth Streak plates
Reasons for performing each transformation step Ca++ O CH2 P Base OH Sugar Transformation solution = CaCl2 pH = 6.1 Positive charge of Ca+2 ions neutralizes negative charge of phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to enter the cells.
Why perform each transformation step? Incubation on ice slows fluid cell membranes Heat-shock increases the permeability of cell membrane to DNA. Increase the bacterial uptake of foreign DNA. Nutrient broth incubation allows beta lactamase expression GFP Cell wall Beta lactamase (ampicillin resistance)
What is nutrient broth or LB broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression carbohydrates amino acids nucleotides salts vitamins
Which colonies will glow? Grow? Glow? Which colonies will glow? Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp LB/Amp/Ara LB
Central Framework of Molecular Biology DNA RNA Protein Trait