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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.
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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
Outline • Overview • Bacteria and Plasmids • Transformation • The pGLO Plasmid • Experimental Procedures • Extension Activities
What is Bacterial Transformation? Taking up of DNA from the environment by bacterial cells
Bacterial Transformation Lab • Only cells which obtained plasmid DNA will grow… and glow • Bacterial Cells and plasmid DNA are mixed • Cells take up plasmid • Cell/DNA mix is plated on nutrient agar with antibiotic
ori bla 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
Chromosomal Bacterial cell Plasmid DNA Chromosomal DNA Bacterial Cells and DNA
bacteria If few cells grow If many cells grow Growth of Bacteria on Plates Agarose in Petri dish = plate Incubate at 37C lawn colonies
Bacterial Cell Chromosomal DNA Plasmids Bacterial Transformation The uptake of DNA
Methods of transformation • Electroporation • Electrical shock makes cell membranes permeable to DNA • Calcium Chloride/Heat Shock • Chemically-competent cells uptake DNA after heat shock
araC ori pGLO GFP bla pGLO Plasmid • bla gene • beta-lactamase enzyme • Ampicillin resistance • GFP gene • Green Fluorescent Protein • Aequorea victoria jellyfish • araC gene • Regulates GFP transcription • ori • Allows plasmid replication
pGLO Plasmid: Most Important Components • bla gene • Bacteria with this gene grow in the presence of ampicillin • GFP gene • Bacteria with this gene glow under near UV light pGLO GFP bla
Transformation Procedures +CaCl2 +CaCl2
Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Reasons for Each Transformation Step • CaCl2 treatment Positive charge of Ca2+ ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids
Reasons for Each Transformation Step • Incubation on iceslows fluid cell membranes • Heat-shockincreases permeability of cell membrane • Nutrient broth incubationallows beta lactamase expression
Transformation Results Lb/amp/ara 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 White – no glow
Transfer Bacteria Glowing Bacteria from Transformation Plate with Arabinose Plate without Arabinose Extension Activity I: Transcriptional Regulation Arabinose controls expression of GFP gene: Incubate overnight @ 37C
Plate with Arabinose Plate without Arabinose Extension Activity I: Transcriptional Regulation arabinose = no glow +arabinose = glow After overnight incubation
araC GFP Gene Arabinose araC GFP Gene araC RNA Polymerase GFP Gene Transcriptional Regulation of GFP by Arabinose araC repressor blocks transcription Arabinose bindsrepressor, 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 42C during the experiment Compare results with number of colonies obtained during the normal protocol
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