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Práctica de Transformación
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Práctica de Transformación

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Transcript

  • 1.
  • 2. pGLO™ Transformation
  • 3. WhyTeachBacterial Transformationand Protein Purification?
    Powerful teaching tool
    Laboratory extensions
    Real-world connections
    Link to careers and industry
    Standards based
  • 4.
  • 5.
  • 6. pGLO™ Bacterial Transformation Kit
    Bio-Rad pGLO Kit Advantages
    Standards-based
    Comprehensive curricula for inquiry-based investigations
    Compatible with 50 minute class periods
    Serves entire class of 32 students (up to 4 students per group)
    Cost-effective
    Success in student’s hands
    Safe
    Striking results!
  • 7. WorkshopTime Line
    • Introduction
    • 8. Transform bacteria with pGLO plasmid
  • DNA
    RNA
    Protein
    Trait
    Central Framework of Molecular Biology
  • 9. En procariontes
    El promotor es la secuencia que señala el comienzo de la transcripción del ADN a ARN, y es por ello el lugar de enlace de la ARN polimerasa. El promotor se encuentra en una de las dos hebras del ADN, y la orientación y posición del promotor dictará cuál de las dos hélices del ADN servirá como molde para sintetizar el ARN.
    Muchos promotores necesitan de proteínas activadoras antes de que puedan unirse eficazmente a su respectiva polimerasa.
    Generalmente, el enlace de la ARN polimerasa con el promotor es lo que facilita a que la hélice de ADN se abra para permitir la subsecuente transcripción a ARN.
  • 10. 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
  • 11.
  • 12. Using GFP as a biological tracer
    http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
    With permission from Marc Zimmer
  • 13. pGLO Bacterial Transformation Kit
  • 14. Transformation
  • 15. Transformation 2
    Day 1
    Day 2
  • 16. E. coli cells
    • sensitive to antibiotics
    • 17. can’t glow
    • 18. competent - able to be transformed
    Bacterial chromosome
  • 19. AmpR
    Ara
    promoter
    Transformation
    Plasmid containing:
    • Ampicillin resistance gene (always expressed)
    • 20. Ara promoter - turned on in the presence of arabinose
  • GFP gene
    Transformation
    Jellyfish DNA
    GFP = Green Fluorescent Protein
    glows under UV light
  • 21. AmpR
    Ara
    GFP
    Transformation
    E. coli cells
    Plasmid
    Jellyfish DNA
  • 22. AmpR
    GROW ON AN AGAR PLATE
    GFP
    Ara
    … that can GLOW!
    Transformation
    Recombinant Bacteria…
  • 23. makes all transformed bacteria resistant to ampicillin
    controls GFP gene expression
    only turned on in the presence of arabinose
    Transformation
    AmpR
    pGLO plasmid
    GFP
    Ara
    promoter
  • 24. What is Transformation?
    GFP
    Uptake of foreign DNA, often a circular plasmid
    Beta-lactamase
    Ampicillin
    Resistance
  • 25. 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
  • 26. Bacterial DNA
    Bacterial cell
    Plasmid DNA
    Genomic DNA
  • 27. The Many Faces of Plasmids
    Graphic representation
    Scanning electron micrograph of supercoiled plasmid
  • 28. GeneExpression
    • Beta Lactamase
    • 29. Ampicillin resistance
    • 30. Green Fluorescent Protein (GFP)
    • 31. Aequorea victoria jellyfish gene
    • 32. araC regulator protein
    • 33. Regulates GFP transcription
  • Bacterial Transformation
    Cell wall
    GFP
    Bacterial
    chromosomal DNA
    Beta lactamase
    (ampicillin resistance)
    pGLO plasmids
  • 34. Transcriptional Regulation
    Lactose operon
    Arabinose operon
    pGLO plasmid
  • 35. ara Operon
    lac Operon
    araC
    B
    A
    D
    LacI
    Z
    Y
    A
    Effector (Arabinose)
    Effector(Lactose)
    araC
    B
    A
    D
    Z
    Y
    A
    LacI
    RNA Polymerase
    RNA Polymerase
    B
    A
    D
    araC
    Z
    Y
    A
    Transcriptional Regulation
  • 36. 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
  • 37. Methods of Transformation
    Electroporation
    Electrical shock makes cell membranes permeable to DNA
    Calcium Chloride/Heat-Shock
    Chemically-competent cells uptake DNA after heat shock
  • 38. Transformation Procedure
    Suspend bacterial colonies in Transformation solution
    Add pGLO plasmid DNA
    Place tubes on ice
    Heat-shock at 42°C and place on ice
    Incubate with nutrient broth
    Streak plates
  • 39. Reasons for Performing Each Transformation Step?
    Ca++
    O
    Ca++
    P
    O
    O
    Base
    O
    O
    CH2
    Sugar
    Transformation solution = CaCI2
    Positive charge of Ca++ ions shields negative
    charge of DNA phosphates
    O
    Ca++
    O
    O
    P
    Base
    O
    O
    CH2
    Sugar
    OH
  • 40. 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)
  • 41. What is Nutrient Broth?
    Luria-Bertani (LB) broth
    Medium that contains nutrients for bacterial growth and gene expression
    Carbohydrates
    Amino acids
    Nucleotides
    Salts
    Vitamins
  • 42. LB/Amp
    LB/Amp/Ara
    LB
    Grow?Glow?
    Follow protocol
    On which plates will colonies grow?
    Which colonies will glow?
  • 43. LaboratoryQuick Guide