This Little Light of Mine: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 HitomiMonte Vista High School, Danville, CA.

Kirk BrownTracy High School, Tracy, CA.

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

Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters

OutlineOutline

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

OverviewOverview

What is Bacterial What is Bacterial Transformation?Transformation?

Taking up of DNA from the environment by bacterial cells

Bacterial Transformation LabBacterial Transformation Lab

• Only cells which obtained plasmid DNA will grow… and glow

• Cell/DNA mix is plated on nutrient agar with antibiotic

• Cells take up plasmid

• Bacterial Cells and plasmid DNA are mixed

Bacteria and PlasmidsBacteria and Plasmids

What is a plasmid?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 DNABacterial Cells and DNA

Chromosomal DNA

Chromosomal

Bacterial cell

Plasmid DNA

Growth of Bacteria Growth of Bacteria on Plateson Plates

Agarose in Petri dish = plate

bacteria

Incubate at 37CIf few

cells growIf many

cells grow

colonies lawn

TransformationTransformation

Bacterial Transformation

Plasmids

Chromosomal DNA

Bacterial Cell

The uptake of DNA

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 PlasmidThe pGLO Plasmid

pGLOori

blaGFP

araC

pGLO Plasmidbla gene

beta-lactamase enzyme Ampicillin resistance

GFP gene Green Fluorescent Protein Aequorea victoria jellyfish

araC gene Regulates GFP transcription

oriAllows plasmid replication

pGLO

blaGFP

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

18

pGlo Plasmid Additional Features

19

1. Arabinose sugar binds regulator.

2. Regulator plus arabinose complex recruits RNA Polymerase to bind promotor.

3. Gene is transcribed into mRNA and “read.”

20

Experimental ProceduresExperimental Procedures

Transformation Procedures

+CaCl2 +CaCl2

Transformation Procedures

Reasons for Each Transformation Step

CaCl2 treatment

Positive charge of Ca2+ ions neutralizes: • negative charge of DNA

phosphates • negative charge of

membrane phospholipids

Ca++

Ca++

OCH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Incubation on ice slows fluid cell membranes

Heat-shock increases permeability of cell membrane

Nutrient broth incubation allows beta lactamase expression

Reasons for Each Transformation Step

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

Lb/amp/ara

White – no glow

Extension ActivitiesExtension Activities

Extension Activity I: Transcriptional Regulation

Arabinose controls expression of GFP gene:

Glowing Bacteria from Transformation

Plate with Arabinose

Plate without Arabinose

Transfer Bacteria

Incubate overnight @ 37C

Extension Activity I: Transcriptional Regulation

−arabinose = no glow +arabinose = glow

Plate with Arabinose

Plate without Arabinose

After overnight incubation

Transcriptional Regulation of GFP by

ArabinosearaC GFP Gene

araC GFP Gene

RNA Polymerase

Arabinose

araC

GFP Gene

araC repressor blocks transcription

Arabinose binds repressor, changing its conformation

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 42C during

the experiment

Compare results with number of colonies obtained during the normal protocol

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