pglo™ transformation. central framework of molecular biology dna rna proteintrait
Post on 18-Dec-2015
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pGLO™ Transformation
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Central Framework of Molecular Biology
DNA RNA Protein Trait
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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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Transformation Procedure Overview
Day 1
Day 2
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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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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
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
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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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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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Transformation Procedure Overview
Day 1
Day 2250ul
1000ul
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