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

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

3. Lab Timeline • Introduction /Transformation • Transform bacteria with pGLO plasmid

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

5. 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

7. 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

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

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

10. 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

11. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

12. The Many Faces of Plasmids Graphic representation Scanning electron micrograph of supercoiled plasmid

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

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

15. Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid

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

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

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

19. Transformation Procedure Overview Day 2 Day 1

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

21. 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)

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

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

24. 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!

25. Volume Measurement

26. 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!!