Pglo Lab Background Information

This Little Light of Mine: Transform bacteria with a Jellyfish gene to make them glow Module based on a kit from Bio-Rad Laboratories, Inc. Adapted by Dan Murray from a presentation by

Stan Hitomi Monte Vista High School, Danville, CA.

Kirk Brown Tracy High School, Tracy, CA.

Aequorea victoria: Source of “glowing gene” for this experiment

Jellyfish Gene put into Other Critters

Outline • • • • • •

Overview Bacteria and Plasmids Transformation The pGLO Plasmid Experimental Procedures Extension Activities

Overview

What is Bacterial Transformation? Taking up of DNA from the environment by bacterial cells

Bacterial Transformation Lab • Bacterial Cells and plasmid DNA are mixed • Cells take up plasmid • Cell/DNA mix is plated on nutrient agar with antibiotic • Only cells which obtained plasmid DNA will grow… and glow

Bacteria and Plasmids

What is a plasmid? Small circular DNA molecule Replicates autonomously Originally evolved in bacteria „ May contain antibiotic resistance gene or be modified to contain other genes „ „ „

bla is an ampicillin resistance gene

„

ori bla

Bacterial Cells and DNA

Chromosomal

Bacterial cell

Plasmid DNA Chromosomal DNA

Growth of Bacteria on Plates bacteria

If few cells grow

colonies

Agarose in Petri dish = plate

Incubate at 37°C

If many cells grow

lawn

Transformation

Bacterial Transformation The uptake of DNA Bacterial Cell Chromosomal DNA

Plasmids

Methods of transformation „ Electroporation „ Electrical

shock makes cell membranes permeable to DNA

„ Calcium

Chloride/Heat Shock

„ Chemically-competent

DNA after heat shock

cells uptake

The pGLO Plasmid

pGLO Plasmid „ bla

gene

„ beta-lactamase „

enzyme

Ampicillin resistance

„ GFP

gene

araC ori

Fluorescent Protein „ Aequorea victoria jellyfish

pGLO

„ Green

„ araC

gene

„ Regulates

GFP transcription

„ori „Allows plasmid replication

bla

GFP

pGLO Plasmid: Most Important Components „

bla gene „

„

Bacteria with this gene grow in the presence of ampicillin

pGLO

GFP gene „

Bacteria with this gene glow under near UV light

bla

GFP

Experimental Procedures

Transformation Procedures

+CaCl2

+CaCl2

Transformation Procedures

Reasons for Each Transformation Step Ca++

nCaCl2 treatment

Ca++

O

O P O

Base

O

Positive charge of Ca2+ ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids

O

CH2

Sugar O

Ca++ O P O

Base

O CH2

O

Sugar

OH

Reasons for Each Transformation Step oIncubation on ice slows fluid cell membranes

pHeat-shock increases permeability of cell membrane

qNutrient broth incubation allows beta lactamase expression

Biotechnology

Explorer Program

Serious About Science Education