transformation procedure overview

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Transformation Procedure Overview. Day 2. Day 1. What is Transformation?. Uptake of foreign DNA, often a circular plasmid. GFP. Beta-lactamase Ampicillin Resistance. Bacterial DNA. Bacterial cell. Plasmid DNA. Genomic DNA. The Many Faces of Plasmids. Graphic representation. - PowerPoint PPT Presentation

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Page 1: Transformation  Procedure Overview
Page 2: Transformation  Procedure Overview

Transformation Procedure Overview

Day 1

Day 2

Page 3: Transformation  Procedure Overview

What is Transformation?

• Uptake of foreign DNA, often a circular plasmid

GFP

Beta-lactamase

Ampicillin

Resistance

Page 4: Transformation  Procedure Overview

Bacterial DNA

Plasmid DNA

Bacterial cell

Genomic DNA

Page 5: Transformation  Procedure Overview

The Many Faces of Plasmids

Scanning electron micrograph of supercoiled plasmid

Graphic representation

Page 6: Transformation  Procedure Overview

GeneExpression

• Beta Lactamase– Ampicillin resistance

• Green Fluorescent Protein (GFP)– Aequorea victoria

jellyfish gene

• araC regulator protein– Regulates GFP

transcription

Page 7: Transformation  Procedure Overview

Bacterial Transformation

Beta lactamase(ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP

Page 8: Transformation  Procedure Overview

Transcriptional Regulation

• Lactose operon

• Arabinose operon

• pGLO plasmid

Page 9: Transformation  Procedure Overview

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

Page 10: Transformation  Procedure Overview

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

Page 11: Transformation  Procedure Overview

Methods of Transformation

• Electroporation– Electrical shock makes cell membranes

permeable to DNA

• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after

heat shock

Page 12: Transformation  Procedure Overview

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

Page 13: Transformation  Procedure Overview

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++

Page 14: Transformation  Procedure Overview

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

Page 15: Transformation  Procedure Overview

What is Nutrient Broth? • Luria-Bertani (LB) broth

• Medium that contains nutrients for bacterial growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins

Page 16: Transformation  Procedure Overview

Grow? Glow?

• Follow protocol

• On which plates will colonies grow?

• Which colonies will glow?