Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

39
Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School

Transcript of Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Page 1: 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

Page 2: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

Page 3: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Central Framework of Molecular Biology

DNA RNA Protein Trait

Page 4: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

The Central Dogma

Trait (phenotype

)

Page 5: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 6: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 7: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Using GFP as a biological tracer

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer

Page 8: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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

Page 9: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

GFP Transformed into Bacteria

Page 10: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Applications of GFP

Page 11: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

GFP in a Plant, Arabadopsis

Page 12: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

GFP in a Mouse

Page 13: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Genetically Engineered Fish

Page 14: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Bacterial DNA

Plasmid DNA

Bacterial cell

Genomic DNA

Page 15: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 16: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 17: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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.

Page 18: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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.

Page 19: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

GeneExpression

• Beta Lactamase– Ampicillin resistance

• Green Fluorescent Protein (GFP)– Aequorea victoria

jellyfish gene

• araC regulator protein– Regulates GFP

transcription

pBAD

Page 20: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

pGLO

Page 21: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Bacterial Transformation

Beta lactamase(ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP

Flagellum

Page 22: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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

Page 23: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Transformation Procedure Overview

Day 1

Day 2

Page 24: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 25: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 26: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Transcriptional Regulation

• Lactose operon

• Arabinose operon

• pGLO plasmid

Page 27: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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

Page 28: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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

Page 29: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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 30: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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 31: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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 32: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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 33: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

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

Page 34: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Grow? Glow?

• Follow protocol

• On which plates will colonies grow?

• Which colonies will glow?

Page 35: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

LaboratoryQuick Guide

Page 36: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Volume Measurement

Page 37: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.

Aequorea victoria- a jellyfish

Page 38: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.
Page 39: Chapter 20 DNA Transformation A. P. Biology Mr. Knowles Liberty Senior High School.