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 Agarose in Petri dish = plate If few cells grow Incubate at 37  C If many cells grow colonies 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 cells uptake DNA after heat shock

The pGLO Plasmid

pGLO Plasmid



bla

gene  beta-lactamase enzyme  Ampicillin resistance 

GFP

gene  Green Fluorescent Protein 

Aequorea victoria

jellyfish 

araC

gene  Regulates GFP transcription 

ori

 Allows plasmid replication

ori bla pGLO araC GFP

pGLO Plasmid: Most Important Components



bla

gene  Bacteria with this gene grow in the presence of ampicillin  GFP gene  Bacteria with this gene glow under near UV light

bla pGLO GFP

Experimental Procedures

Transformation Procedures +CaCl 2 +CaCl 2

Transformation Procedures

Reasons for Each Transformation Step



CaCl 2 treatment

Positive charge of Ca

2+

ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids

Ca ++ Ca ++

O O P O CH 2 O

O Sugar Base Ca ++

O O P O O CH 2

O Sugar Base

OH

Reasons for Each Transformation Step



Incubation on ice

slows fluid cell membranes



Heat-shock

increases permeability of cell membrane



Nutrient broth incubation

allows beta lactamase expression

Transformation Results

Only cells getting pGLO plasmid grow and glow All cells grow since there is no antibiotic on the plate Without pGLO plasmid, nothing can grow All cells grow since there is no antibiotic on the plate

Extension Activities

Extension Activity I:

Transcriptional Regulation

Arabinose controls expression of GFP gene:

Transfer Bacteria

Glowing Bacteria from Transformation Plate with Arabinose

Incubate overnight @ 37



C

Plate without Arabinose

Extension Activity I:

Transcriptional Regulation

 arabinose = no glow +arabinose = glow

After overnight incubation

Plate with Arabinose Plate without Arabinose

araC

Transcriptional Regulation of GFP by Arabinose

araC GFP Gene

araC

repressor blocks transcription Arabinose araC GFP Gene Arabinose binds repressor, changing its conformation RNA Polymerase GFP Gene Altered repressor leaves DNA, RNA polymerase can perform transcription

Extension Activity II:

Tweaking the Transformation Protocol

Test effect of various components of the transformation protocol:   plate ampicillin concentration plate arabinose concentration    amount of plasmid DNA used in the experiment amount of cells used in the experiment length of time cells/DNA mix is kept at 42  C during the experiment Compare results with number of colonies obtained during the normal protocol

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