Nucleic Acids and Genetics Chapter 5 A. P. Biology Mr. Knowles Liberty Senior High School.
Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
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Transcript of Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Chapter 20 DNA Transformation
A. P. Biology
Mr. Knowles
Liberty Senior High School
pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
Central Framework of Molecular Biology
DNA RNA Protein Trait
The Central Dogma
Trait (phenotype
)
Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
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
Real-World Applications
GFP Transformed into Bacteria
Applications of GFP
GFP in a Plant, Arabadopsis
GFP in a Mouse
Genetically Engineered Fish
Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
What is a plasmid?
• An extrachromosomal, circular piece of autonomously replicating DNA.
• Originally evolved by bacteria.
• May express an antibiotic resistance gene
or be modified to express proteins of interest.
The Many Faces of Plasmids
Scanning electron micrograph of supercoiled plasmid
Graphic representation
• Plasmids act as vectors – carriers for foreign DNA fragments (genes) that have been inserted into them and then propagated.
• Usually put into cells to make the desired protein.
GeneExpression
• Beta Lactamase– Ampicillin resistance
• Green Fluorescent Protein (GFP)– Aequorea victoria
jellyfish gene
• araC regulator protein– Regulates GFP
transcription
pBAD
pGLO
Bacterial Transformation
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
Flagellum
What is Transformation?
• Uptake of foreign DNA, often a circular plasmid.
• May involve bacteria or many eukaryotic types of cells.
• Process of copying genes of interest and making proteins from them.
GFP
Beta-lactamase
Ampicillin
Resistance
Transformation Procedure Overview
Day 1
Day 2
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
pBAD
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
pBAD
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
• We will have THREE Types of Agar Plates:- LB only- LB + Amp- LB + Amp + Arabinose
Grow? Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
LaboratoryQuick Guide
Volume Measurement
Aequorea victoria- a jellyfish