Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

• Summer 2008 Workshop

• in Biology and Multimedia

• for High School Teachers

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

AP Biology Lab 6:Genetic Engineering

viaBacterial Transformation

Making E. coli glow like jellyfish

Amy Dickson, Prospect Hill Academy Charter School

All images by Christine Rodriguez and Amy Dickson

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

WHY SHOULD WE DO THIS?

Genetic Engineering is now widely used:

• Bacteria that produce human insulin

• Corn that produces insecticide

• Rice that produces extra vitamin A

• Goats that produce spider silk

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

WHY SHOULD WE DO THIS?

To SEE the Central Dogma in action:

DNA

RNA

Protein

Trait

GFP Gene

• found in jellyfish

• engineered into bacteria

Green Fluorescent Protein

GLOWING CELLS

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

QUICK REVIEW

Promoter -

Plasmid -

Transformation - a process in which bacteria take up DNA from their environment

a small, circular piece of bacterial DNA that is not part of the chromosome

an “on/off” switch for a gene

- can be triggered by electric shock or heat shock

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

STARTING MATERIALS

Bacterial chromosome

E. coli cells

• sensitive to antibiotics

• can’t glow

• competent - able to be transformed

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

STARTING MATERIALS

AmpR

Ara

promoter

Plasmid containing:

• Ampicillin resistance gene (always expressed)

• Ara promoter - turned on in the presence of

arabinose

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

STARTING MATERIALS

GFP gene

Jellyfish DNA

GFP = Green Fluorescent Protein

glows under UV light

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

GFP

Jellyfish DNA

STARTING MATERIALS

E. coli cells

AmpR

Ara

Plasmid

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

END RESULT

AmpR

Ara

GFP

Recombinant Bacteria…

… that can GLOW!

GROW ON AN AGAR PLATE

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

HOWEVER…things are actually a bit more

complex.

AmpR

Arapromoter

GFP

pGLO plasmid

makes all transformed bacteria resistant to ampicillin

controls GFP gene expression

only turned on in the presence of arabinose

Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard

College.

YOUR TASK:Design an experimental procedure for genetically engineering glowing

bacteria.

pGLO

Goals to consider:

#1 - Make recombinant bacteria

#2 - Select for only the recombinant bacteria

#4 - Establish a control for your experiment to demonstrate that it’s the plasmid that causes ampicillin resistance and the ability to glow.

#3 - Make the recombinant bacteria glow