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Biology 11H

Biology 11H. pGLO Prep: Gene Transformation in Bacteria. Objectives. By the end of this lab you should be able to: Describe the function of plasmids in bacteria Relate genes to protein synthesis. Key Terms!. Plasmid Transformation Gene Protein DNA Agar. Lab Skills & Processes :.

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Biology 11H

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  1. Biology 11H pGLO Prep: Gene Transformation in Bacteria

  2. Objectives By the end of this lab you should be able to: • Describe the function of plasmids in bacteria • Relate genes to protein synthesis

  3. Key Terms! • Plasmid • Transformation • Gene • Protein • DNA • Agar

  4. Lab Skills & Processes: • Sterile technique • Proper pipet use • Transferring bacterial colonies • DNA transfer (transformation) • Heat Shock • Incubation • Gene regulation

  5. What are we ACTUALLY doing? • Transfer the pGLO plasmid (which contains the GFP gene and the Bla gene) into e. coli bacteria • Grow the transformed e. coli bacteria on different agar media (LB, LB/amp, LB/amp/ara,) GFP: Green Fluorescent Protein (which glows bright green when exposed to UV light) Bla:beta lactamase (provides resistance to the antibiotic ampicillin) LB: nutrient media (named after Luria and Bertani) that provides the bacteria with the required nutrients for survival amp: short for ampicillin ara: short for arabinose

  6. ACK!!! e.coli!??!?! • The e.coli used is strain HB101 K-12 • The one that gives you food poisoning is strain O157 H7

  7. Some Details: • Arabinose is a sugar that provides energy and acts as a C source for bacteria (chemoheterotrophs!) • The genes required to break down arabinose are not turned on unless arabinose is present SOOOOOOO: • The pGLO plasmid won’t be turned on unless arabinose is present in the agar plates (LB/amp/ara) 

  8. More Details: • Ampicillin is an antibiotic that inhibits peptidoglycan formation in the cell wall • The Bla gene on the pGLO plasmid produces a protein that inactivates ampicillan SOOOOO: • If the Bla gene is successfully transformed into the bacteria then they will survive on the ampicillin infused agar plates (LB/amp)  BUT: what other gene is also on the pGLO plasmid that is required to activate it?

  9. Get Ready!! • Next class we will be: • Analysing the original “untransformed” e.coli • Transferring the pGLO plasmid into the e.coli • Growing the e.coli on the 3 agar mediums (LB; LB/amp; LB/amp/ara)

  10. Lab Info: • Sterile technique: minimize contamination • How to choose a colony (round) and transfer from starter plate to suspension (no agar) • DNA transfer: “soapy” looking loop

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