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 via Bacterial 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: DN A RN A ProteinTrait 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 Ara promoter 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