práctica de transformación
TRANSCRIPT
pGLO™ Transformation
Why Teach
Bacterial Transformationand Protein Purification?
• Powerful teaching tool
• Laboratory extensions
• Real-world connections
• Link to careers and industry
• Standards based
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!
WorkshopTime Line
• Introduction
• Transform bacteria with pGLO plasmid
Central Framework of Molecular Biology
DNA RNA Protein Trait
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.
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
Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
pGLO Bacterial Transformation Kit
Transformation
Transformation 2
Day 1
Day 2
Bacterial chromosome
E. coli cells
• sensitive to antibiotics
• can’t glow
• competent - able to be transformed
Transformation
AmpR
Ara
promoter
Plasmid containing:
• Ampicillin resistance gene (always expressed)
• Ara promoter - turned on in the presence of
arabinose
Transformation
GFP gene
Jellyfish DNA
GFP = Green Fluorescent Protein
glows under UV light
Transformation
GFP
Jellyfish DNA
E. coli cells
AmpR
Ara
Plasmid
Transformation
AmpR
Ara
GFP
Recombinant Bacteria…
… that can GLOW!
GROW ON AN AGAR PLATE
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
What is Transformation?
• Uptake of foreign DNA, often a circular plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
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
Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
The Many Faces of Plasmids
Scanning electron micrograph of supercoiled plasmid
Graphic representation
GeneExpression
• Beta Lactamase– Ampicillin resistance
• Green Fluorescent Protein (GFP)– Aequorea victoria
jellyfish gene
• araC regulator protein– Regulates GFP
transcription
Bacterial Transformation
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
Transcriptional Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
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
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
Methods of Transformation
• Electroporation– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after
heat shock
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
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++
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
What is Nutrient Broth?
• Luria-Bertani (LB) broth
• Medium that contains nutrients for bacterial growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins
Grow? Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
LaboratoryQuick Guide