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Biorad pGlo-Biotechnology

Biorad pGlo-Biotechnology. Biotechnology. Explorer Program. Serious About Science Education. Molecular Biology Lab 6. Bacterial Transformation p GLO. Links to real world. study of biological processes (ex. biosynthesis of proteins) localization of gene expression cell movement

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Biorad pGlo-Biotechnology

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  1. Biorad pGlo-Biotechnology Biotechnology Explorer Program Serious About Science Education Helwig

  2. Molecular Biology Lab 6 Bacterial Transformation p GLO Helwig

  3. Links to real world • study of biological processes (ex. biosynthesis of proteins) • localization of gene expression • cell movement • cell fate during development • formation of different organs • screen-able marker to identify transgenic organisms Helwig

  4. Green Fluorescent Protein Aequorea victoria Helwig

  5. What makes jelly fish glow? What makes jelly fish glow? Calcium: the body's conductor Why can photo proteins, the flash-bulb proteins that jellies such as Aequorea Victoria turn on to give the outer edge of the creature's bell-shaped body a bluish or greenish glow? The answer centers on calcium. The jelly's light switch turns on in the presence of calcium ions, and the calcium ion is one of nature's favorite intercellular messengers, Whenever you tense a muscle or waggle a finger; whenever your heart pounds out a beat, pulsing blood through a tangle of veins and arteries, calcium has prompted the muscle to act, contract or twitch. Helwig

  6. Alba the GFP Bunny Alba the GFP Bunny Helwig

  7. What is transformation? • Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance Helwig

  8. What is a plasmid? ori bla • A circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance gene or • be modified to express proteins of interest Helwig

  9. pGlo plasmid araC ori pGLO GFP bla • Beta Lactamase • Ampicillin resistanceThe beta lactamase protein is produced and secreted by bacteria that contain the plasmid. Inactivates ampicillin. • Green Fluorescent Protein • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription Helwig

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

  11. Bacterial DNA Genomic Bacterialcell PlasmidDNA Genomic DNA Helwig

  12. Transcription regulation araC ori pGLO GFP bla • Lactose operon • Arabinose operon • pGLO plasmid Helwig

  13. ARABINOSE • SOURCE OF ENERGY AND CARBON FOR BACTERIA. FOUND IN PLANTS AND IS NORMALLY USED AS A SOURCE FOOD FOR BACTERIA. • THE BACTERIAL GENES THAT MAKE DIGESTIVE ENZYMES TO BREAK DOWN ARABINOSE FOR FOOD ARE NOT EXPRESSED WHEN ARABINOSE IS ABSENT. • WHEN ARABINOSE IS PRESENT GENES TURN ON. WHEN ABSENT GENES TURN OFF. • ARABINOSE INITIATES TRANSCRIPTION OF THESE GENES BY PROMOTING THE BINDING OF RNA POLYMERASE. Helwig

  14. ARABINOSE OPERON Helwig

  15. Expression of Green Fluorescent Protein Helwig

  16. Ara Operon and ara GFP Operon ara GFP Operon araC GFP Gene araC GFP Gene RNA Polymerase araC GFP Gene ara Operon araC B A D Effector(Arabinose) Effector(Arabinose) araC B A D RNA Polymerase araC B A D Helwig

  17. lac Operon and ara operon lac Operon ara Operon araC LacI Z Y A B A D Effector (Lactose) Effector (Arabinose) araC B A D LacI Z Y A RNA Polymerase B A D Z Y A araC RNA Polymerase Helwig

  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 Helwig

  19. Transformation procedure • Suspend bacterial colonies in Transformation Solution • Add pGLO plasmid DNA • Place tubes on ice • Heat shock at 42oC and place on ice • Incubate withnutrient broth • Streak plates Helwig

  20. Reasons for performing each transformation step Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH • Transformation solution = CaCl2 • pH = 6.1 Positive charge of Ca+2 ions neutralizes negative charge of phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to enter the cells. Helwig

  21. Why perform each transformation step? Cell wall GFP Beta lactamase (ampicillin resistance) • Incubation on iceslows fluid cell membranes • Heat-shockincreases the permeability of cell membrane to DNA. Increase the bacterial uptake of foreign DNA. • Nutrient broth incubationallows beta lactamase expression Helwig

  22. What is nutrient broth or LB broth? What is nutrient broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • carbohydrates • amino acids • nucleotides • salts • vitamins Helwig

  23. RECOVERY Recovery is the 10 minute incubation period after you added the LB which allows the cells to grow and express the ampicillin resistant beta galactamase so the newly transformed cells survive on ampicllin plates. Helwig

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

  25. How do I measure 250 micro liters using the pipette? Volume Measurement Helwig

  26. Central Framework of Molecular Biology DNA RNA Protein Trait Helwig

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