Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP) Central Framework of Molecular Biology DNA RNA Protein Trait 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 GFP Transformed into Bacteria Applications of GFP GFP in a Plant, Arabadopsis GFP in a Mouse Genetically Engineered Fish 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 Gene Expression • Beta Lactamase – Ampicillin resistance • Green Fluorescent Protein (GFP) – Aequorea victoria jellyfish gene • araC regulator protein – Regulates GFP transcription pBAD pGLO 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. GFP • May involve bacteria or many eukaryotic types of cells. • Process of copying genes of interest and making proteins from them. Beta-lactamase Ampicillin Resistance Transformation Procedure Overview Day 1 Day 2 Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid Transcriptional Regulation ara Operon lac Operon LacI Z Y A araC B Effector (Arabinose) Effector (Lactose) LacI Z Y A araC Y A B A D RNA Polymerase RNA Polymerase Z A D araC pBAD B A D Gene Regulation ara GFP Operon ara Operon ara C B A D araC GFP Gene Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC B A D GFP Gene RNA Polymerase araC pBAD GFP Gene 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++ Ca++ O O P O O CH2 Base O Sugar 1. Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O Base O CH2 O 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 Grow? Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow? Laboratory Quick Guide Volume Measurement Aequorea victoria- a jellyfish