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pGLO Transformation LAB AP LAB 6

araC. ori. pGLO. GFP. bla. pGLO Transformation LAB AP LAB 6. BIO-RAD lab book. http://www.mshri.on.ca/nagy/GFP%20mice.jpg. Aequorea victoria : Source of “glowing gene” for this experiment. Jellyfish Gene put into Other Critters. http://www.lafuga.de/GFP_pig.jpg.

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pGLO Transformation LAB AP LAB 6

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  1. araC ori pGLO GFP bla pGLO Transformation LABAP LAB 6 BIO-RAD lab book http://www.mshri.on.ca/nagy/GFP%20mice.jpg

  2. Aequorea victoria: Source of “glowing gene” for this experiment

  3. Jellyfish Gene put into Other Critters http://www.lafuga.de/GFP_pig.jpg http://www.technologyreview.com/files/21291/monkey_x600.jpg

  4. PLASMID Extrachromosomal DNA Often carry genes for antibiotic resistance Can be passed from one bacterium to another http://www.agen.ufl.edu/~owens/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpg

  5. Bacterial Cell Chromosomal DNA Plasmids Bacterial Transformation The uptake of DNA

  6. araC ori pGLO GFP bla pGLO plasmid ARABINOSE OPERON (INDUCIBLE) Turns on when arabinose sugar is present Allows bacteria to digest this sugar Ori- Plasmid Replication genes GFP-Green Fluorescent Protein - Glows green in fluorescent light bla (beta-lactamase) - On all time - Makes protein that breaks down ampicillin - Provides ampicillin resistance

  7. ARABINOSE OPERON REGULATION ara Operon INDUCIBLE OPERON PRESENCE OF ARABINOSE TURNS ON GENES TO MAKE ENZYMES TO DIGEST ARABINOSE araC B A D Effector (Arabinose) araC B A D RNA Polymerase araC B A D

  8. pGLO Regulation ara Operon GFP GENE HAS BEEN ADDED TO ara OPERON WHEN ARABINOSE IS PRESENT, OPERON IS TURNED ON andGFP GENEIS EXPRESSED TOO Cells “glow” on mediawith arabinose araC B A D GFP Gene Effector (Arabinose) GFP Gene araC B A D RNA Polymerase araC B A D GFP Gene

  9. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Reasons for Each Transformation Step • CaCl2 treatment Positive charge of Ca2+ ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids

  10. Reasons for Each Transformation Step • Incubation on iceslows fluid cell membranes • Heat-shockincreases permeability of cell membrane • Nutrient broth incubationallows beta lactamase expression

  11. Selection for plasmid uptake • Antibiotic becomes a selecting agent • only bacteria with the plasmid will grow on antibiotic (ampicillin) plate only transformedbacteria grow all bacteria grow a a a a a a a a a a a a a a a a a LB plate LB/amp plate cloning

  12. Transformation Results LB PLATELuria Broth + - PGLO = NO Plasmid → All cells grow since there is no antibiotic on the plate

  13. Transformation Results LB/AMP PLATELuria Broth with antibiotic + - PGLO = NO plasmid → NO GROWTHCells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.

  14. Transformation Results LB/AMP PLATELuria Broth with antibiotic + + PGLO = Plasmid added → LAWNCells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

  15. Transformation Results Cells with pGLO plasmid GROW & GLOW-can grow on media with antibiotic GLOW on media with arabinose (turns on GFP gene) LB/AMP/ARA PLATELuria Broth+ antibiotic|+ arabinose + + PGLO = Plasmid added →

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