pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

Lab Timeline Introduction /Transformation Transform bacteria with pGLO plasmid When a cell takes up and expresses a new piece of trait: Uses: plants to be resistent to pest Bacterial to digest oil spills, Gene therapy

Central Framework of Molecular Biology: The Central Dogma DNA RNA Protein Trait

Context Genetic engineering of organism Use of experimental controls Interpretation of experimental results Calculate transformation efficiency GMO Cell biology Evolution: antibiotic resistance, selection mechanism, adaptation Genetics: Central Dogma, gene regulation, lac operon

GRP (green fluorescent protein from jelly fish!)

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

Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

What is Transformation? GFP Uptake of foreign DNA, often a circular plasmid Beta-lactamase Ampicillin Resistance

What is a plasmid? A circular piece of autonomously replicating DNA Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest

Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

The Many Faces of Plasmids 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

Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

Transcriptional Regulation Lactose operon Arabinose operon pGLO plasmid

Transcriptional Regulation RNA Polymerase Z Y A LacI Effector (Lactose) lac Operon B A D araC RNA Polymerase Effector (Arabinose) ara Operon

Gene Regulation B A D ara Operon ara GFP Operon GFP Gene araC RNA Polymerase Effector (Arabinose) ara Operon RNA Polymerase araC ara GFP Operon GFP Gene Effector (Arabinose)

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 Overview Day 1 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 Day 2

Reasons for Performing Each Transformation Step? Ca++ O Ca++ O P O Base O O CH2 Sugar Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O 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

Grow? Glow? Follow protocol On which plates will colonies grow? LB/Amp Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp/Ara LB

Lab Safety The E. coli is not pathogenic Safety Procedure: Decontaminate work surface each day All liquid or solid waste needs to be decontaminated (using bleach!) Wash hands after handling bacteria and before leaving lab Carefully following all protocol / procedure. If you are allergic to ampicillin…let me know! UV lamp do not stare into the light or shine on your own skin!

Volume Measurement

Lab technique Sterile technique! Do not introduce contaminating bacteria The inoculation loops, pipets, agar plates should NOT touch or be placed onto contaminating surfaces! Wash your Hands!! Be careful with timing…it’s the most important part of the lab!!