Download - Práctica de Transformación
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pGLO™ Transformation
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Why Teach
Bacterial Transformationand Protein Purification?
• Powerful teaching tool
• Laboratory extensions
• Real-world connections
• Link to careers and industry
• Standards based
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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!
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WorkshopTime Line
• Introduction
• Transform bacteria with pGLO plasmid
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Central Framework of Molecular Biology
DNA RNA Protein Trait
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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.
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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
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Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
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pGLO Bacterial Transformation Kit
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Transformation
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Transformation 2
Day 1
Day 2
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Bacterial chromosome
E. coli cells
• sensitive to antibiotics
• can’t glow
• competent - able to be transformed
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Transformation
AmpR
Ara
promoter
Plasmid containing:
• Ampicillin resistance gene (always expressed)
• Ara promoter - turned on in the presence of
arabinose
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Transformation
GFP gene
Jellyfish DNA
GFP = Green Fluorescent Protein
glows under UV light
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Transformation
GFP
Jellyfish DNA
E. coli cells
AmpR
Ara
Plasmid
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Transformation
AmpR
Ara
GFP
Recombinant Bacteria…
… that can GLOW!
GROW ON AN AGAR PLATE
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Transformation
AmpR
Arapromoter
GFP
pGLO plasmid
makes all transformed bacteria resistant to ampicillin
controls GFP gene expression
only turned on in the presence of arabinose
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What is Transformation?
• Uptake of foreign DNA, often a circular plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
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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
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Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
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The Many Faces of Plasmids
Scanning electron micrograph of supercoiled plasmid
Graphic representation
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GeneExpression
• Beta Lactamase– Ampicillin resistance
• Green Fluorescent Protein (GFP)– Aequorea victoria
jellyfish gene
• araC regulator protein– Regulates GFP
transcription
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Bacterial Transformation
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
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Transcriptional Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
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Transcriptional Regulation
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA Polymerase
Z Y A
Z Y ALacI
Effector (Lactose)
Z Y ALacI
lac Operon
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Gene Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
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Methods of Transformation
• Electroporation– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after
heat shock
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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
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Reasons for Performing Each Transformation Step?
1. Transformation solution = CaCI2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++
Ca++
OCH2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
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Why Perform Each Transformation Step?
2. Incubate on iceslows fluid cell membrane
3. Heat-shockIncreases permeability of membranes
4. Nutrient broth incubationAllows beta-lactamase expression
Beta-lactamase(ampicillin resistance)
Cell wall
GFP
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What is Nutrient Broth?
• Luria-Bertani (LB) broth
• Medium that contains nutrients for bacterial growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins
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Grow? Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
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LaboratoryQuick Guide