práctica de transformación

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