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• 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 GFP Gene RN A • found in jellyfish Protein Green Fluorescent Protein Trait GLOWING CELLS • engineered into bacteria Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. QUICK REVIEW Promoter Plasmid - an “on/off” switch for a gene a small, circular piece of bacterial DNA that is not part of the chromosome Transformation - a process in which bacteria take up DNA from their environment - can be triggered by electric shock or heat shock Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. STARTING MATERIALS E. coli cells • sensitive to antibiotics • can’t glow Bacterial chromosome • competent - able to be transformed Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. STARTING MATERIALS 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 Jellyfish DNA GFP gene GFP = Green Fluorescent Protein glows under UV light Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. STARTING MATERIALS E. coli cells Plasmid GFP Jellyfish DNA Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. END RESULT GROW ON AN AGAR PLATE Recombinant Bacteria… … that can GLOW! Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College. HOWEVER… things are actually a bit more complex. makes all transformed bacteria resistant to ampicillin pGLO plasmid 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. Goals to consider: #1 - Make recombinant bacteria #2 - Select for only the recombinant bacteria #3 - Make the recombinant bacteria glow #4 - Establish a control for your experiment to demonstrate that it’s the plasmid that causes ampicillin resistance and the ability to glow. Life Sciences-HHMI Outreach. Copyright 2008 President and Fellows of Harvard College.