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Transformation Phase I Training BACTERIAL TRANSFORMATION

Transformation Phase I Training BACTERIAL TRANSFORMATION . Fluorescent Protein Activities. Bacterial Transformation Protein Purification. Scorpion- Natural Light. Scorpion- UV Light. Bioluminescence vs. Fluorescence. Bioluminescence. Fluorescence.

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Transformation Phase I Training BACTERIAL TRANSFORMATION

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  1. Transformation Phase I TrainingBACTERIAL TRANSFORMATION

  2. Fluorescent Protein Activities Bacterial Transformation Protein Purification

  3. Scorpion- Natural Light Scorpion- UV Light Bioluminescence vs. Fluorescence Bioluminescence Fluorescence http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/ Natural Light In the Dark A fluorescent organism absorbs light at one wavelength (UV) and a re-emits the light at a visible wavelength= color Bioluminescent organism produces its own light.

  4. Many organisms have the ability to fluoresce Mushroom coral www.worldnetcams.com/sealife/cerianthus.jpg Jellyfish Amphipod Spider’s palps

  5. Discovery of GFP-1960’s Aequorea victoria OSAMU SHIMOMURA Co-winner of Nobel Prize

  6. 17 Mut mgrape 1 DsRed.T1 6 Mut 33 Mut Dimer 2 6 Mut mCherry mRFP1 3 Mut 8 Mut 3 Mut mHoneydew 4 Mut 7 Mut mStrawberry mBanana mTangerine mOrange Roger Tsien and Rainbow Proteins

  7. Frog Egg Extract + sperm DNA A. Desai Fly Embryo T. Megraw Frog Cell C.E. Walczak Human Cell J. Waters Frog Egg Extract + DNA-coated beads R. Heald Marsupial Cell S.L. Kline Worm Embryo I.M. Cheeseman MITOSIS a similar process in diverse species ……...using various organisms to understand humans:

  8. Fluorescent Proteins-Applications Co-localization Richard Klemke Lab, UCSD Professor Dept. of Pathology & Moores Cancer Center - Model for cellular migration - Localization within a cell relates to function - Lasp-1 protein modified with GFP - vinculin antibodies stain vinculin protein red - overlap is yellow; implying an interaction - arrow indicates direction of migration http://klemkelab.ucsd.edu/index.html • GFP [27kD, 238 amino acids] • Lasp-1 [261 amino acids] • - involved in actin cytoskeleton organization • vinculin [117kD, 1066 amino acids] • - part of a complex that connects to molecules that adhere

  9. Fluorescent Proteins-Applications Zebrafish Richard Klemke Lab, UCSD Professor Dept. of Pathology & Moores Cancer Center - Model of tumor invasion and angiogensis - GFP transgenic zebrafish, vertebrate model - dsRed expressing human tumor cells - blue control cells - 3D microscopy http://klemkelab.ucsd.edu/research/zebrafish.html

  10. Fluorescent Proteins-Applications Hollis Cline Lab, Scripps Research Institute Professor Dept. of Pathology & Moores Cancer Center - Testing how experience alters neuron development - uses albino Xenopus tadpoles and Zebrafish http://www.scripps.edu/cb/cline/research.html

  11. What is a plasmid? promoter • A small circular piece of DNA • Naturally occurring • Can be altered in lab to express protein of interest GFP Origin Stop AmpR PM1PM2 Green Cherry Blue Tangerine Grape Yellow

  12. FP transformation procedure

  13. E. coli

  14. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

  15. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

  16. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days on plate with ampicillin. Bacterial chromosome

  17. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days on plate with ampicillin. Bacterial chromosome Bacteria now express cloned fluorescent protein…

  18. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH How does the plasmid get in? • CaCl2 • Positive charge of Ca++ ions shields negative charge of DNA phosphates

  19. How does the plasmid get in? • Incubate on ice slows fluid cell membrane • Heat-shock increases permeability of membranes • Leave in heat 45 seconds!!! • Too short, and bacteria won't let in plasmid. • Too long, and the bacteria will die.

  20. How are plasmids engineered? DNA Plasmid Vector Host DNA fragments (i.e. coral or jellyfish FP coding DNA) Ligate (paste) fragments into cut DNA vector Cut plasmids open with restriction enzymes + Cut genomic DNA into fragments Screen for and select plasmid containing FP gene

  21. B-lactamase Plasmid Details promoter is constitutive (always on) codes for GFP protein and has a HIS-tag for protein purification (utilized in BioBridge’s Protein Purification Kit) promoter GFP Origin Stop AmpR Ampicillin resistance is coded by B-lactamase, which destroys ampicillin’s structure/function. LB Only LB Amp transpeptidase transpeptidase Amp peptidoglycan layer peptidoglycan layer survives death (unless AmpR)

  22. The rainbow of mFruit Fluorescent Proteins

  23. CA Science Content Standards Investigation & Experimentation Scientific progress is made by asking meaningful questions and conducting careful investigations. Students will: … identify and communicate sources of unavoidable experimental error. … identify possible reasons for inconsistent results, such as sources of error or uncontrolled conditions. … formulate explanations by using logic and evidence.

  24. CA Science Content Standards Biology Genetics The genetic composition of cells can be altered by incorporation of exogenous DNA into the cells. … the general structures and functions of DNA, RNA, and protein. … genetic engineering (biotechnology) is used to produce novel biomedical and agricultural products. … basic DNA technology (transformation) is used to construct recombinant DNA molecules. … exogenous DNA can be inserted into bacterial cells to alter their genetic makeup and support expression of new protein products.

  25. CA Science Content Standards Biology Cell Biology … the central dogma of molecular biology outlines the flow of information from transcription of ribonucleic acid (RNA) in the nucleus to translation of proteins on ribosomes in the cytoplasm. … cells are enclosed within semi permeable membranes that regulate their interaction with their surroundings. … prokaryotic cells, eukaryotic cells (including those from plants and animals), and viruses differ in complexity and general structure.

  26. CA Science Content Standards Biology Evolution … students know why natural selection acts on the phenotype rather than the genotype of an organism … students know new mutations are constantly being generated in a gene pool … students know how natural selection determines the differential survival of groups of organisms … students know a great diversity of species increases the chance that at least some organisms survive major changes in the environment

  27. CA Science Content Standards Biology Ecology … stability in an ecosystem is a balance between competing effects. As a basis for understanding this concept …students know bio diversity is the sum total of different kinds of organisms and is affected by alterations of habitats

  28. DNA Transformation by BioBridge Online resources: http://biobridge.ucsd.edu/

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