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This Little Light of Mine: Transform bacteria with a Jellyfish gene to make them glow Module based on a kit from Bio-Rad Laboratories, Inc. Adapted by Dan Murray from a presentation by Stan Hitomi Monte Vista High School, Danville, CA. Kirk Brown Tracy High School, Tracy, CA. Aequorea victoria: Source of “glowing gene” for this experiment Jellyfish Gene put into Other Critters Outline • • • • • • Overview Bacteria and Plasmids Transformation The pGLO Plasmid Experimental Procedures Extension Activities Overview What is Bacterial Transformation? Taking up of DNA from the environment by bacterial cells Bacterial Transformation Lab • Bacterial Cells and plasmid DNA are mixed • Cells take up plasmid • Cell/DNA mix is plated on nutrient agar with antibiotic • Only cells which obtained plasmid DNA will grow… and glow Bacteria and Plasmids What is a plasmid? Small circular DNA molecule Replicates autonomously Originally evolved in bacteria May contain antibiotic resistance gene or be modified to contain other genes bla is an ampicillin resistance gene ori bla Bacterial Cells and DNA Chromosomal Bacterial cell Plasmid DNA Chromosomal DNA Growth of Bacteria on Plates bacteria If few cells grow colonies Agarose in Petri dish = plate Incubate at 37°C If many cells grow lawn Transformation Bacterial Transformation The uptake of DNA Bacterial Cell Chromosomal DNA Plasmids Methods of transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat Shock Chemically-competent DNA after heat shock cells uptake The pGLO Plasmid pGLO Plasmid bla gene beta-lactamase enzyme Ampicillin resistance GFP gene araC ori Fluorescent Protein Aequorea victoria jellyfish pGLO Green araC gene Regulates GFP transcription ori Allows plasmid replication bla GFP pGLO Plasmid: Most Important Components bla gene Bacteria with this gene grow in the presence of ampicillin pGLO GFP gene Bacteria with this gene glow under near UV light bla GFP Experimental Procedures Transformation Procedures +CaCl2 +CaCl2 Transformation Procedures Reasons for Each Transformation Step Ca++ nCaCl2 treatment Ca++ O O P O Base O Positive charge of Ca2+ ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids O CH2 Sugar O Ca++ O P O Base O CH2 O Sugar OH Reasons for Each Transformation Step oIncubation on ice slows fluid cell membranes pHeat-shock increases permeability of cell membrane qNutrient broth incubation allows beta lactamase expression Transformation Results Only cells getting pGLO plasmid grow and glow All cells grow since there is no antibiotic on the plate Without pGLO plasmid, nothing can grow All cells grow since there is no antibiotic on the plate Extension Activities Extension Activity I: Transcriptional Regulation Arabinose controls expression of GFP gene: Transfer Bacteria Glowing Bacteria from Transformation Plate with Arabinose Incubate overnight @ 37°C Plate without Arabinose Extension Activity I: Transcriptional Regulation −arabinose = no glow +arabinose = glow After overnight incubation Plate with Arabinose Plate without Arabinose Transcriptional Regulation of GFP by Arabinose araC GFP Gene araC repressor blocks transcription Arabinose araC araC GFP Gene RNA Polymerase GFP Gene Arabinose binds repressor, changing its conformation Altered repressor leaves DNA, RNA polymerase can perform transcription Extension Activity II: Tweaking the Transformation Protocol Test effect of various components of the transformation protocol: plate ampicillin concentration plate arabinose concentration amount of plasmid DNA used in the experiment amount of cells used in the experiment length of time cells/DNA mix is kept at 42°C during the experiment Compare results with number of colonies obtained during the normal protocol Biotechnology Explorer Program Serious About Science Education
