pGLO and Ampicillin Resistance Transformation of E. coli Inspire your students – tomorrow’s researchers CCTCA February 12, 2016

Ampicillin resistance and pGLO™ Transformation Bob Malyk BSc. , MEd Ampicillin resistance and pGLO™ Transformation Bob Malyk BSc., MEd. biobob4@hotmail.com Biology Teacher (retired) Ridley College St. Catharines, ON Canada Bio-Rad (Canada) Life Science Education – Biotechnology Explorer Consultant

GFP Workshop Timeline Introduction Transform bacteria with pGLO plasmid

Why Teach Bacterial Transformation and Protein Purification? Powerful teaching tool Meet national science standards Laboratory extensions Real-world connections Link to careers and industry

Central Framework of Molecular Biology DNA RNA Protein Trait

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

pGLO™ Bacterial Transformation Kit Bio-Rad pGLO Kit Advantages Standards-based Comprehensive curricula for inquiry-based investigations Compatible with 50 minute class periods Serves entire class of 32 students (up to 4 students per group) Cost effective $120.80 ($3.78/student) Success in student’s hands Safe Striking results! pGLO™ Bacterial Transformation Kit

Transformation Procedure Suspend bacterial colonies in Transformation solution Add pGLO plasmid DNA Place tubes in ice Heat-shock at 42°C and place on ice Incubate with nutrient broth Streak plates

Transformation Procedure Day 1 Day 2

Volume Measurement

pGLO Bacterial Transformation Kit

What is Transformation? GFP Beta-lactamase Ampicillin Resistance 1928 Frederick Griffith 1944 Avery and McLeod Uptake of foreign DNA, often a circular plasmid

What is Transformation? F. Griffith 1928

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

The Many Faces of Plasmids Transmission electron micrograph Graphic representation Agarose gel

Protein Size 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

Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

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

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