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Producing a Strain of E.coli that Glows in the Dark

Producing a Strain of E.coli that Glows in the Dark. Exercise 10. Announcements. Post Lab 10 and Pre Lab 11 is due by your next lab LNA Glow Lab is assigned today, and is due next week in lab Exam II is during your lab period the week of May 5 and will cover Exercises 8- 11

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Producing a Strain of E.coli that Glows in the Dark

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  1. Producing a Strain of E.colithat Glows in the Dark Exercise 10

  2. Announcements • Post Lab 10 and Pre Lab 11 is due by your next lab • LNA Glow Lab is assigned today, and is due next week in lab • Exam II is during your lab period the week of May 5 and will cover Exercises 8-11 • The Final Exam is scheduled for Monday, May 12 from 8:00-11:00 AM.Locations TBA. If this conflicts with any of your other final exams, you must fill out the Final Exam Conflict Request Form by 5 PM on 5/7/14.

  3. Goals • To create an ampicillin-resistant population of E. coli by introducing a plasmid that contains an ampicillin resistance gene • Understand the lux Operon and how it is used to create luminescent bacteria • Understand the pUC18 plasmid and how it serves as a control in this experiment

  4. Penicillin • One of the most important anti-infective agents used in clinical medicine • Interferes with bacterial cell wall synthesis • Over 500 semi-synthetic penicillins have been made during the past 30 years

  5. Plasmids • Plasmids are small circular DNA molecules that exist apart from the chromosomes in most bacterial species • Many contain genes that enable bacteria to survive and prosper in certain environments b/c they carry genes that provide resistance to antibiotics

  6. pUC18 Plasmid • Circular DNA that contains 2686 nucleotides (very small) • Contains an ampicillin-resistance gene

  7. pUC18 Plasmid

  8. Transformation Process of introducing plasmids into living bacterial cells. • Procedure: • Treat bacteria with calcium chloride • Add the plasmid DNA • Select for the transformed bacteria on antibiotic media

  9. lux Operon • Contains two genes that code for luciferase and several genes that code for enzymes which produce luciferins • The lux Operon. The lux operon contains two genes for the luciferase enzyme (A and B). This enzyme is composed of two different polypeptide chains. The operon also contains several other genes (R, T, C, D, E, G, And Rib) that are thought to code for enzymes which produce the substrates for the light-emitting reaction. These substrates are called luciferins and are long chain fatty aldehydes.

  10. The Exercise • Part I: Preparation of Competent Cells • Review procedure on page 122 of your lab manual. This has been done for you prior to starting the experiment. • Part II: Uptake of Plasmid by competent cells • Part III: Selection on ampicillin plates • Follow procedure in your lab manual. Note that to store your plates in the dark, you will put them in a paper sack and then in a box designated for your section

  11. Results Part IV:Examination of cultures in the dark • You will have to attend open hours on:

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