Ch. 5A: Transforming Bacteria with Recombinant Plasmids

1. Ch. 5A: Transforming Bacteria with Recombinant Plasmids

2. Describe the role of transformation in the gene cloning process • Explain the purpose of each control in the transformation experiment • Explain how the information encoded in a gene is expressed as a trait Learning goals

3. A recombinant plasmid must be taken up by bacteria bacteria cell machinery used to replicate and to express the gene of interest. If transformed with the pARA-R plasmid bacteria can be identified Ampicillin will prevent the growth of cells that do not carry an ampicillin resistance gene Arabinose will activate the bacteria promoter that controls expression of the rfpgene. Key Ideas

4. Go topg 76 and complete Lab 5A Transforming Bacteria with the pARA-R Plasmid

5. araC gene PBAD rfp gene mRNA araC protein RFP expression Biotech Experience Transcription Translation

6. 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 Biotech Experience araC protein

7. araC gene PBAD rfp gene RFP (red fluorescent protein) mRNA Transcription RFP expression Biotech Experience 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

8. 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 Plating Tips

9. Agar is like 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 More Plating Tips

10. 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 Remind Students

11. 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 Sterile technique

12. 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

13. “oops” plates

15. 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

16. 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?