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AP Bio: Wednesday, 2.16.11 Planning Lab 6A; Mutations

In this AP Biology lab, students explore the fascinating process of genetic engineering by designing a procedure to transform E. coli bacteria to express Green Fluorescent Protein (GFP), enabling them to glow like jellyfish. The lab focuses on understanding gene regulation, including the use of promoters and the "on/off" switch mechanism to control GFP expression in bacteria. Students will work in groups to create flowcharts, perform a computer-based mutations lab, and complete the transformation experiment, all while reinforcing their knowledge of molecular genetics.

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AP Bio: Wednesday, 2.16.11 Planning Lab 6A; Mutations

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  1. AP Bio: Wednesday, 2.16.11Planning Lab 6A; Mutations • Homework: • Study for Test on Molecular Genetics • Do Now: • Get a copy of Lab 6A: Transformation • Today’s Goals: • Design a procedure for genetically engineering E. coli cells to glow. • Describe the effects of different types of mutations. • Agenda: • Mini-Lecture: Intro to Lab 6A – Transformation • Explanation of Lab Report • In Lab Groups: (1) Use the colored cards to make a flow chart and then a procedural list for completing this lab. (2) Complete the Computer-Based Mutations Lab. Print and hand to me when finished.

  2. AP Biology Lab 6:Genetic EngineeringviaBacterial Transformation Making E. coli glow like jellyfish Amy Dickson, Prospect Hill Academy Charter School All images by Christine Rodriguez and Amy Dickson

  3. GOALS OF THIS LAB PROJECT: • Make E. coli bacteria glow like jellyfish • By inserting the GFP (green fluorescent protein) gene from a jellyfish into a bacterial plasmid • Control when the bacteria express this protein • By connecting the GFP gene to an “on/off” switch that causes it to be expressed only in certain environments

  4. 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

  5. DNA RNA Protein Trait Green Fluorescent Protein • GFP Gene • found in jellyfish • engineered into bacteria GLOWING CELLS WHY SHOULD WE DO THIS? To SEE the Central Dogma in action:

  6. DNA RNA Protein Trait WHY SHOULD WE DO THIS? To SEE the Central Dogma in action: X on Trait To understand how gene expression is regulated - how cells (and the scientists who manipulate them) control when genes are turned on/off.

  7. A BIT OF BACKGROUND ON GENE REGULATION • Promoter: • A short DNA sequence upstream of a gene where RNA pol. binds to start transcription • Serves as the on/off switch for the gene  blocking it turns the gene off • Why do this? • Making proteins only when needed saves energy and materials

  8. A BIT OF BACKGROUND ON GENE REGULATION • Example: • Arabinose is a sugar that bacteria can digest • But no need to make enzymes unless arabinose is around • Normal condition: Promoter blocked by Ara repressor • In presence of arabinose: repressor is inactivated; gene is turned on genes for arabinose-digesting enzymes promoter genes not expressed Ara repressor (active) Arabinose sugar binding site genes expressed! Arabinose sugar (inducer)

  9. QUICK REVIEW Promoter - Plasmid - Transformation - an “on/off” switch for a gene a small, circular piece of bacterial DNA that is not part of the chromosome a process in which bacteria take up DNA from their environment - can be triggered by electric shock or heat shock

  10. STARTING MATERIALS E. coli cells • sensitive to antibiotics • can’t glow • competent - able to be transformed Bacterial chromosome

  11. AmpR Ara promoter STARTING MATERIALS • Plasmid containing: • Ampicillin resistance gene (always expressed) • Ara promoter - turned on in the presence of arabinose

  12. GFP gene STARTING MATERIALS Jellyfish DNA GFP = Green Fluorescent Protein glows under UV light

  13. AmpR Ara GFP STARTING MATERIALS E. coli cells Plasmid Jellyfish DNA

  14. AmpR GROW ON AN AGAR PLATE GFP Ara … that can GLOW! END RESULT Recombinant Bacteria…

  15. makes all transformed bacteria resistant to ampicillin controls GFP gene expression only turned on in the presence of arabinose HOWEVER… things are actually a bit more complex. AmpR pGLO plasmid GFP Ara promoter

  16. pGLO 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 only when we want them to. #4 - Establish a control for your experiment to demonstrate that it’s the plasmid that causes ampicillin resistance and the ability to glow.

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