pGLO™ Transformation and Purification of  Green Fluorescent Protein (GFP)
<ul><li>Stan Hitomi </li></ul><ul><li>Coordinator – Math & Science </li></ul><ul><li>Principal – Alamo School </li></ul><u...
Why   Teach Bacterial Transformation and Protein Purification? <ul><li>Powerful teaching tool </li></ul><ul><li>Laboratory...
 
pGLO ™  Bacterial Transformation Kit <ul><li>Bio-Rad pGLO Kit Advantages </li></ul><ul><li>Standards-based </li></ul><ul><...
Green Fluorescent Protein (GFP) Chromatography Kit <ul><li>GFP Purification Kit Advantages </li></ul><ul><li>Cloning in ac...
Workshop Time Line <ul><li>Introduction </li></ul><ul><li>Transform bacteria with pGLO plasmid </li></ul>
Central Framework of Molecular Biology DNA RNA   Protein Trait
Links to  Real-world <ul><li>GFP is a visual marker </li></ul><ul><li>Study of biological processes  (example: synthesis o...
 
Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
pGLO Bacterial Transformation Kit
Transformation  Procedure Overview <ul><li>Day 2 </li></ul>Day 1 10
What is Transformation?   <ul><li>Uptake of foreign DNA,  often a circular plasmid </li></ul>GFP Beta-lactamase Ampicillin...
What is a plasmid?   <ul><li>A circular piece of autonomously replicating DNA </li></ul><ul><li>Originally evolved by bact...
Bacterial DNA   Plasmid DNA Bacterial cell Genomic DNA
The Many Faces of Plasmids   Scanning electron micrograph of supercoiled plasmid Graphic representation
Gene Expression   <ul><li>Beta Lactamase </li></ul><ul><ul><li>Ampicillin resistance </li></ul></ul><ul><li>Green Fluoresc...
Bacterial Transformation Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial  chromosomal DNA Cell wall GFP
Transcriptional Regulation   <ul><li>Lactose operon </li></ul><ul><li>Arabinose operon </li></ul><ul><li>pGLO plasmid </li...
Transcriptional Regulation   B A D araC B A D araC RNA Polymerase Effector  (Arabinose) araC B A D ara Operon RNA Polymera...
Gene Regulation   RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector   (Arabinose) B A D ara...
Methods of Transformation <ul><li>Electroporation </li></ul><ul><ul><li>Electrical shock makes cell membranes permeable to...
Transformation Procedure <ul><li>Suspend bacterial colonies in Transformation solution </li></ul><ul><li>Add pGLO plasmid ...
Reasons for Performing Each Transformation Step?   <ul><li>Transformation solution = CaCI 2 </li></ul><ul><ul><li>Positive...
Why Perform Each Transformation Step? 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of...
What is Nutrient Broth?  <ul><li>L uria- B ertani (LB) broth </li></ul><ul><li>Medium that contains nutrients for bacteria...
Grow?   Glow?   <ul><li>Follow protocol </li></ul><ul><li>On which plates will colonies  grow ? </li></ul><ul><li>Which co...
Laboratory Quick Guide
Volume Measurement
Webinars <ul><li>Enzyme Kinetics — A Biofuels Case Study </li></ul><ul><li>Real-Time PCR — What You Need To Know and Why Y...
Upcoming SlideShare
Loading in...5
×

pGLO Tutorial - English

1,746
-1

Published on

Are you interested in the pGLO™ experiment? Learn more about this hands-on laboratory activity with a FREE download of BioRad’s pGLO™ experiment power point.

Published in: Education, Technology
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,746
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
13
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

pGLO Tutorial - English

  1. 2. pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
  2. 3. <ul><li>Stan Hitomi </li></ul><ul><li>Coordinator – Math & Science </li></ul><ul><li>Principal – Alamo School </li></ul><ul><li>San Ramon Valley Unified School District </li></ul><ul><li>Danville, CA </li></ul><ul><li>Kirk Brown </li></ul><ul><li>Lead Instructor, Edward Teller Education Center </li></ul><ul><li>Science Chair, Tracy High School </li></ul><ul><li>and Delta College, Tracy, CA </li></ul><ul><li>Bio-Rad Curriculum and Training Specialists: </li></ul><ul><li>Sherri Andrews, Ph.D. </li></ul><ul><li>[email_address] </li></ul><ul><li>Essy Levy, M.Sc. </li></ul><ul><li>[email_address] </li></ul><ul><li>Leigh Brown, M.A. </li></ul><ul><li>[email_address] </li></ul>Instructors
  3. 4. Why Teach Bacterial Transformation and Protein Purification? <ul><li>Powerful teaching tool </li></ul><ul><li>Laboratory extensions </li></ul><ul><li>Real-world connections </li></ul><ul><li>Link to careers and industry </li></ul><ul><li>Standards based </li></ul>
  4. 6. pGLO ™ Bacterial Transformation Kit <ul><li>Bio-Rad pGLO Kit Advantages </li></ul><ul><li>Standards-based </li></ul><ul><li>Comprehensive curricula for inquiry-based investigations </li></ul><ul><li>Compatible with 50 minute class periods </li></ul><ul><li>Serves entire class of 32 students (up to 4 students per group) </li></ul><ul><li>Cost-effective </li></ul><ul><li>Success in student’s hands </li></ul><ul><li>Safe </li></ul><ul><li>Striking results! </li></ul>
  5. 7. Green Fluorescent Protein (GFP) Chromatography Kit <ul><li>GFP Purification Kit Advantages </li></ul><ul><li>Cloning in action </li></ul><ul><li>Links to biomanufacturing </li></ul><ul><li>Biopharmaceutical development </li></ul><ul><li>Amazing visual results </li></ul>
  6. 8. Workshop Time Line <ul><li>Introduction </li></ul><ul><li>Transform bacteria with pGLO plasmid </li></ul>
  7. 9. Central Framework of Molecular Biology DNA RNA Protein Trait
  8. 10. Links to Real-world <ul><li>GFP is a visual marker </li></ul><ul><li>Study of biological processes (example: synthesis of proteins) </li></ul><ul><li>Localization and regulation of gene expression </li></ul><ul><li>Cell movement </li></ul><ul><li>Cell fate during development </li></ul><ul><li>Formation of different organs </li></ul><ul><li>Screenable marker to identify transgenic organisms </li></ul>
  9. 12. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer
  10. 13. pGLO Bacterial Transformation Kit
  11. 14. Transformation Procedure Overview <ul><li>Day 2 </li></ul>Day 1 10
  12. 15. What is Transformation? <ul><li>Uptake of foreign DNA, often a circular plasmid </li></ul>GFP Beta-lactamase Ampicillin Resistance
  13. 16. What is a plasmid? <ul><li>A circular piece of autonomously replicating DNA </li></ul><ul><li>Originally evolved by bacteria </li></ul><ul><li>May express antibiotic resistance gene </li></ul><ul><li>or be modified to express proteins of interest </li></ul>
  14. 17. Bacterial DNA Plasmid DNA Bacterial cell Genomic DNA
  15. 18. The Many Faces of Plasmids Scanning electron micrograph of supercoiled plasmid Graphic representation
  16. 19. Gene Expression <ul><li>Beta Lactamase </li></ul><ul><ul><li>Ampicillin resistance </li></ul></ul><ul><li>Green Fluorescent Protein (GFP) </li></ul><ul><ul><li>Aequorea victoria jellyfish gene </li></ul></ul><ul><li>araC regulator protein </li></ul><ul><ul><li>Regulates GFP transcription </li></ul></ul>
  17. 20. Bacterial Transformation Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial chromosomal DNA Cell wall GFP
  18. 21. Transcriptional Regulation <ul><li>Lactose operon </li></ul><ul><li>Arabinose operon </li></ul><ul><li>pGLO plasmid </li></ul>
  19. 22. Transcriptional Regulation B A D araC B A D araC RNA Polymerase Effector (Arabinose) araC B A D ara Operon RNA Polymerase Z Y A Z Y A LacI Effector (Lactose) Z Y A LacI lac Operon
  20. 23. Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) B A D araC B A D araC RNA Polymerase Effector (Arabinose) araC B A D ara Operon
  21. 24. Methods of Transformation <ul><li>Electroporation </li></ul><ul><ul><li>Electrical shock makes cell membranes permeable to DNA </li></ul></ul><ul><li>Calcium Chloride/Heat-Shock </li></ul><ul><ul><li>Chemically-competent cells uptake DNA after heat shock </li></ul></ul>
  22. 25. Transformation Procedure <ul><li>Suspend bacterial colonies in Transformation solution </li></ul><ul><li>Add pGLO plasmid DNA </li></ul><ul><li>Place tubes on ice </li></ul><ul><li>Heat-shock at 42°C and place on ice </li></ul><ul><li>Incubate with nutrient broth </li></ul><ul><li>Streak plates </li></ul>
  23. 26. Reasons for Performing Each Transformation Step? <ul><li>Transformation solution = CaCI 2 </li></ul><ul><ul><li>Positive charge of Ca ++ ions shields negative </li></ul></ul><ul><ul><li>charge of DNA phosphates </li></ul></ul>Ca ++ Ca ++ O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar Sugar O Ca ++
  24. 27. 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 Beta-lactamase (ampicillin resistance) Cell wall GFP
  25. 28. What is Nutrient Broth? <ul><li>L uria- B ertani (LB) broth </li></ul><ul><li>Medium that contains nutrients for bacterial growth and gene expression </li></ul><ul><ul><li>Carbohydrates </li></ul></ul><ul><ul><li>Amino acids </li></ul></ul><ul><ul><li>Nucleotides </li></ul></ul><ul><ul><li>Salts </li></ul></ul><ul><ul><li>Vitamins </li></ul></ul>
  26. 29. Grow? Glow? <ul><li>Follow protocol </li></ul><ul><li>On which plates will colonies grow ? </li></ul><ul><li>Which colonies will glow ? </li></ul>LB/Amp LB/Amp/Ara LB
  27. 30. Laboratory Quick Guide
  28. 31. Volume Measurement
  29. 32. Webinars <ul><li>Enzyme Kinetics — A Biofuels Case Study </li></ul><ul><li>Real-Time PCR — What You Need To Know and Why You Should Teach It! </li></ul><ul><li>Proteins — Where DNA Takes on Form and Function </li></ul><ul><li>From plants to sequence: a six week college biology lab course </li></ul><ul><li>From singleplex to multiplex: making the most out of your realtime experiments </li></ul><ul><li>explorer.bio-rad.com  Support  Webinars </li></ul>
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×