pGLO NotesBiology III Spring 2012

Introduction to Transformation Gene: piece of DNA which provides the

instructions for making a protein Genetic Transformation: change

caused by genes. Changes the organism’s traits

Caused by the insertion of genes from different organisms (both same and different species)

Bacterial Transformation

Plasmids

Chromosomal DNA

Bacterial Cell

The uptake of DNA

PLASMIDExtrachromosomal DNA

Often carry genes for antibiotic resistance

Can be passed from one bacterium to another

http://www.agen.ufl.edu/~owens/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpg

Central Framework of Molecular Biology

DNA RNA Protein Trait

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

pGLO Transformation Lab For this lab:

Transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). Source: Bioluminescent jellyfish

Aequorea Victoria Causes jellyfish to glow in the dark under ultra

violet light

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

About the lab… Learn about the process of moving

genes from one organism to another. Transfer plasmids containing the GFP

gene into Ecoli. Bio-Rad’s unique pGLO plasmid encodes

the gene for the GFP and a gene for resistance to antibiotic ampicillin

Contains a gene regulation system

Gene Regulation Organisms regulate expression of their

genes and proteins present in the cells Allows for adaptation to the environment Eliminate wasteful protein production Ex. Ecoli and Catabolism (breaking

down) of arabinose (sugar) Genes involved in breaking down sugar

are not expressed when no sugar is present How is this so???

Regulation of Genes Regulation of the expression of proteins:

Occurs at the level of transcription from DNA into RNA

Takes place at a promoter Promoter=where transcription begins

pGLO plasmid

bla (beta-lactamase)- On all time- Makes protein that breaks down ampicillin- Provides ampicillin resistance

GFP-Green Fluorescent Protein- Glows green in fluorescent light

ARABINOSE OPERON(INDUCIBLE)Turns on when arabinose sugar is presentAllows bacteria to digest this sugar

pGLOori

blaGFP

araCOri-PlasmidReplicationgenes

Ecoli Plasmid

ori

araC

araD

araA

araB

Arabinose

Operon Complex

• RNA Polymerase must bind to the promoter site and continue past the operator site to transcribe mRNA

Arabinose Operon

RNA Polymerase

B A DaraC

Effector (Arabinose)

araC B A D

Promotor (PBAD)

DNA binding Protein: Represses Transcription

Genes coding for digestive enzymes

B A DaraC

Transcription

Ara-GFP Operon

RNA Polymerase

araC GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

Promotor (PBAD)

Transcription

GFP only produced in the presence of Arabinose

Genes coding for digestive enzymes have been replaced by the GFP gene: no metabolism of arabinose

How Does it GLOW?

Unique 3-D Structure of GFP

Resonates when exposed to ultraviolet light

Gives off energy in the form of green fluorescent light

Learn more about GFP:http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

How does it work?

GFP

Beta lactamase(ampicillin resistance)

pGlo Plasmids

Bacterial chromosomal DNA

Cell wall

Transform bacteria with the pGlo plasmid and grow under various conditions

Methods of transformation

DNA molecules are too large to easily diffuse or be transported though the cell membrane

ElectroporationElectrical shock makes cell

membranes permeable to DNACalcium Chloride/Heat Shock

Chemically-competent cells uptake DNA after heat shock

Electroporation

Transformation

Transformation Procedure: Overview

Suspend bacterial colonies in

Transformation Solution Add pGLO plasmid DNA Place tubes on ice

Heat shock at 42oC and place on ice

Incubate with LB nutrient broth

Streak plates

Why perform each step?

CaCl2 treatment on ice crytallizes fluid membranes and stabilizes distribution of charged molecules

CaCl2 Transformation solution provides Ca++

cations that neutralize the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane, allowing the DNA to enter the cells

Ca++

Ca++

OCH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Picture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Why perform each step?

Heat-shock increases permeability of cell membrane

Luria-Bertani Nutrient broth incubation allows beta lactamase expression

Beta lactamase(ampicillin resistance)pGlo

Plasmids

Bacterial chromosomal DNA

Cell wall

Gene selection

Grow transformed bacteria and control bacteria under various conditions.

On which plates will colonies grow?Which colonies will glow?

The pGlo System

Timing is important…be efficient!!

Mix contents before pipetting!!!

A film of plasmid must be on the loop!

Sterile Technique

Bacteria are UBIQUITOUS…they are found EVERYWHERE!

Sterile technique refers to procedures that reduce the possibility of contamination…these techniques protect YOU, your CULTURES and REAGENTS, and LAB EQUIPMENT

pGlo Lab Considerations

Teacher Considerations

Work surfaces Hands Glassware Agar Petri plates Inoculation loops Solutions/Cultures Pipettes

Student Considerations

Work surfaces Hands E.coli starter plates Assorted agar plates Inoculation loops Solutions/Test tubes Pipettes

Closing Considerations

ALWAYS decontaminate you work surfaces with a disinfecting solution: 20ml of liquid household bleach in 1L of tap water. Spray the solution on work surfaces and let

stand for 2 minutes and wipe away ALWAYS thoroughly scrub hands for at

least 20 seconds with soap and hot water before leaving the lab area

Volume Measurement