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

Instructors Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Damon Tighe damon_tighe@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

Why Teach Bacterial Transformation Real-world connections Link to careers and industry Standards based Integration with AP Big Idea 3: Molecular Genetics Options for student inquiry based activities

Technology Engineering Math Science Inquiry

pGLO™ Bacterial Transformation Kit Bio-Rad pGLO Kit Advantages Everything you need comes in the box Compatible with 50 minute class periods Serves entire class of 32 students (up to 4 students per group) Cost-effective Success in student’s hands Safe Striking results!

Workshop Time Line Introduction Transform bacteria with pGLO plasmid Expected results Extensions

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

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

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

pGlo Plasmid Beta Lactamase Ampicillin resistance Green Fluorescent Protein (GFP) Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription

What is Transformation? E. coli cell What is Transformation? Uptake of foreign DNA, often a circular plasmid

Transformation Procedure Overview Day 1 10 Day 2

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

Find your quick guide

Let’s get started! At your station you should have: Ice cup Foam float with 2 microfuge tubes (with 250ul of transformation solution) Marker Microfuge tube labeled “LB” 4 plates taped together Plate labeled “LB” Label one microfuge tube ‘+’ and the other one ‘-’ Go on to step 4 of the Quick Guide

Adding bacteria and plasmid 4. Remove colony from the starter plate and add to the ‘+’ tube. Mix thoroughly. Repeat for the ‘-’ tube. 5. Add 10ul (or one loopful) of plasmid to the ‘+’ tube only 6. Place on ice for 10 minutes

Transformation E. coli Ca+2 Transformation solution of CaCl2. Ca+2 shields negative charge of DNA phosphates Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Ca+2 heat

Transformation Regulation Without Arabinose AraC RNA Pol Beta-lactamase

Transformation Regulation Arabinose operon X RNA Pol

Transformation Regulation With Arabinose – induction of GFP RNA Pol

Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock

Why perform 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? E. coli Why perform each transformation step? 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression

Heat shock 8. Carefully take your ice cup to the water bath. Heat shock cells by placing the float in the water bath for 50 seconds Return to ice for 2 minutes

Volume Measurement

Recovery 9. Add 250ul of LB to each tube. Leave at room temperature for 10 minutes

What is Nutrient Broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins

Preparation for plating 7. Label plates as shown below (write on the bottom of the plates, not the lid). Add your initials to each plate. Save your tape!

Grow? Glow? On which plates will colonies grow? Which colonies will glow?

Questions to consider: Student Inquiry Questions to consider: How important is each step in the lab protocol? What part of the protocol can I manipulate to see a change in the results? Ampicillin concentration Arabinose concentration / timing Heat shock temperature or time Time on ice before and after heat shock Amount of plasmid Amount of bacteria Phase of bacteria used for transformation How do I insure the change I make is what actually affected the outcome? Importance of controlling other variables Collaborative approach / share data Write protocol, get approval, and do it!

More Advanced Questions Student Inquiry More Advanced Questions Are satellite colonies also transformed? What other genes might the pGLO plasmid contain? Can I map the plasmid? Can I remove the pGLO gene? Can I remove the regulation of GFP so I don’t need to add arabinose? Can I detect the presence of the GFP gene using PCR?

Student Inquiry Teacher Considerations What materials and equipment do I have on hand, and what will I need to order? Extra plates, LB, agar, plasmid, ampicillin, arabinose? Incubator, water bath (different temps) Other supplies depending on student questions Consider buying extras in bulk or as refills – many have 1 year + shelf life. What additional prep work will I need? Order supplies Pour plates (different media? different amounts?) Make starter plates (will you need transformed bacteria?) How much time do I want to allow? Limited time? Have students read lab and come up with inquiry questions and protocol before they start. Collaborative approach. Will you need multiple lab periods? Will everyone need the same amount of time?

Transformation Procedure Plating Bacteria New pipet! 10. Put 100 ul of solution onto the appropriate plates 11. Streak plates 12. Stack / tape plates and place in incubator New loop!

pGLO Plasmid pGLO Plasmid DNA

Extensions: Green Fluorescent Protein (GFP) Chromatography Kit 1660005EDU GFP Purification Kit Advantages Links to biomanufacturing Biopharmaceutical development Amazing visual results

Extensions: pGLO Kit SDS-PAGE extension 1660013EDU pGLO SDS-PAGE Kit advantages Learn Protein Electrophoresis Visualize GFP on SDS-PAGE gel, it glows still! Extensions: pGLO Kit SDS-PAGE extension 1660013EDU