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Lab 5a Transformation of Escherichia coli with pARA-R

Lab 5a Transformation of Escherichia coli with pARA-R. “ transformed cell ” – cell has acquired new characteristics “ characteristics ” – due to the expression of incorporated foreign genetic material. The “ acid test ” – does it work?. The Big Picture. 2005 Pearson Education, Inc.

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Lab 5a Transformation of Escherichia coli with pARA-R

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  1. Lab 5a Transformation of Escherichia coli with pARA-R

  2. “transformed cell” – cell has acquired new characteristics “characteristics” – due to the expression of incorporated foreign genetic material The “acid test” – does it work?

  3. The Big Picture 2005 Pearson Education, Inc.

  4. Gene expression – process by which the information encoded in a gene is converted into an observable phenotype • Gene regulation – control mechanisms that turn genes on or off • Inducible proteins – synthesis is regulated depending on the bacterium’s nutritional status • Thank you Francois Jacob and Jacques Monod! • Prokaryote operon model of gene control • Repressors and activators are “trans-acting” – affect expression of their genes no matter on which DNA molecule in the cell these are located. Differential gene expression

  5. Why don’t we see the red protein in any LB growth media? • Cells conserve energy and resources • The rfp gene requires a specific substrate (arabinose) to be turned on (expressed) Why do we need arabinose?

  6. pARA-R construct Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

  7. araC gene PBAD rfp gene mRNA araC protein RFP expression Transcription Translation

  8. araC protein prevents RFP transcription by causing a loop to form in the region of the fp gene r araC gene PBAD rfp gene RFP expression araC protein

  9. Rfp gene has replaced the araA, araB, and araD genes in the normal arabinose operon. These produce enzymes that digest arabinose. araC gene PBAD rfp gene RFP expression araC protein araB araD araA

  10. araC gene PBAD rfp gene RFP (red fluorescent protein) mRNA Transcription RFP expression Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). arabinose Translation RNA polymerase arabinose – araC protein complex araC protein

  11. 1. Plasmid with gene of interest has been produced – confirmed by gel electrophoresis What do we want to know? • 2. Can the plasmid (vector) be taken in by a host cell (E. coli)? • 3. Can the host cell express the gene of interest and produce a product that can be utilized? What do we know?

  12. “Materials” changes for “a” strand folks: Instead of ‘E. Coli plate’ you will use: 100 uL of competent cells Instead of “1 mL 50 mM CaCl2”you will use: 350 uL of LB broth (sterile) How is Lab 5a different from Lab 5?

  13. Note the plate markings: I=LB, II=LB/amp, III=LB/amp/ara Label the bottom of the plate near the edge Open the plates like clam shells Sample goes on the agar, not the lid (really, you need to remind them) Agar Plate tips to tell the students:

  14. Agar is like finger jello, firm but not invincible, be gentle – the “spreader is not a shovel Turn the plates upside down (lids down) for incubation, stacked and taped together After incubation, do not open plates, observe through the bottom

  15. 1. Sterile technique • Using bacteria • Contamination may affect results • 2. Carefully READ and FOLLOW the lab protocol. • Be sure lab partners communicate • 3. No Food or Drinks Three Important aspects to stress with your students

  16. Always follow the protocol carefully – know what you’re doing • Work quickly – less time = less opportunities for contamination • Do not leave any container (tube, plate) open any longer than needed • Watch what your equipment touches – there is no “5 second rule” here. • All tips, tubes and spreaders go in the “contaminated waste” container at the end of the lab. Sterile technique tips for students

  17. Lab 5 day 2 • DO NOT open plates, observe by viewing through the bottoms • Used plates – dispose in the “contaminated waste” bags P- P+ P- P+ P+ LB LB/amp LB/amp/ara

  18. “oops” plates

  19. Some cells without antibiotic resistance do become "freeloaders" and survive because other cells are doing the work of destroying the antibiotic in their immediate vicinity on the plate. They only develop with antibiotics such as ampicillin, that are destroyed by enzymes such as beta lactamase outside of the cell. Satellite colonies

  20. The ampicillin plate is old (meaning that the antibiotic is partially degraded) The transformed cells are plated at very high density (meaning that the plate is covered with huge number of cells) The copy number of the plasmid in the cells is so high that beta lactamase is secreted at high levels, The colonies grow on the plate for several days (allowing more time for degradation). Why do we get satellite colonies?

  21. Probably not, provided that the colony of interest is subsequently picked and grown in fresh medium containing antibiotics. Are satellites a problem?

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