1. Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. Summer 2008 Workshop in Biology and Multimedia for High School Teachers

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. GFP Jellyfish DNA STARTING MATERIALS E. coli cells AmpR Ara Plasmid

10. 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

11. 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

12. 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