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

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

3. GFP Workshop Timeline Introduction
Transform bacteria with pGLO plasmid

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

5. Central Framework of Molecular Biology
DNA
RNA
Protein
Trait

6. 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

7. 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 $ ($3.78/student)
Success in student’s hands
Safe
Striking results!
pGLO™ Bacterial Transformation Kit

8. 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

9. Transformation Procedure
Day 1
Day 2

10. Volume Measurement

11. pGLO Bacterial Transformation Kit

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

13. What is Transformation? F. Griffith 1928

14. 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

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

16. Protein Size Beta Lactamase Ampicillin resistance
Green Fluorescent Protein (GFP)
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription

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

18. Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA

19. Transcriptional Regulation
Lactose operon
Arabinose operon
pGLO plasmid

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

21. 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)

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

23. 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

24. 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)

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

26. 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