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Chapter 20 DNA Transformation. A. P. Biology Mr. Knowles Liberty Senior High School. pGLO ™ Transformation and Purification of Green Fluorescent Protein (GFP). DNA. RNA. Protein. Trait. Central Framework of Molecular Biology. The Central Dogma. Trait (phenotype).
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Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School
pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
DNA RNA Protein Trait Central Framework of Molecular Biology
The Central Dogma Trait (phenotype)
Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
Real-World Applications Links to Real-world • GFP is a visual marker • Study of biological processes (example: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms
Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA
What is a plasmid? • An extrachromosomal, circular piece of autonomously replicating DNA. • Originally evolved by bacteria. • May express an antibiotic resistance gene or be modified to express proteins of interest.
The Many Faces of Plasmids • Plasmids act as vectors – carriers for foreign DNA fragments (genes) that have been inserted into them and then propagated. • Usually put into cells to make the desired protein. Graphic representation Scanning electron micrograph of supercoiled plasmid
GeneExpression • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein (GFP) • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription pBAD
Bacterial Transformation Cell wall GFP Flagellum Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids
What is Transformation? • Uptake of foreign DNA, often a circular plasmid. • May involve bacteria or many eukaryotic types of cells. • Process of copying genes of interest and making proteins from them. GFP Beta-lactamase Ampicillin Resistance
Transformation Procedure Overview Day 2 Day 1
Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid
ara Operon lac Operon araC B A D LacI Z Y A Effector (Arabinose) Effector(Lactose) araC B A D LacI Z Y A RNA Polymerase RNA Polymerase B A D araC Z Y A Transcriptional Regulation pBAD
ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation pBAD
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 42°C and place on ice • Incubate with nutrient broth • Streak plates
Reasons for Performing Each Transformation Step? Ca++ O Ca++ O P O Base • Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH
Why Perform Each Transformation Step? Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance)
What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins • We will have THREE Types of Agar Plates: • LB only • LB + Amp • LB + Amp + Arabinose
LB/Amp LB/Amp/Ara LB Grow?Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow?
