Transcript Slide 1
Ch. 5A: Transforming Bacteria with Recombinant Plasmids Learning goals 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 Key Ideas 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 rfp gene. Go to pg 76 and complete Lab 5A Transforming Bacteria with the pARA-R Plasmid RFP expression Biotech Experience araC gene PBAD Transcription mRNA Translation araC protein rfp gene RFP expression Biotech Experience araC protein prevents RFP transcription by causing a loop to form in the region of the r fp gene araC gene araC protein PBAD rfp gene RFP expression Biotech Experience arabinose Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). RFP (red fluorescent protein) Translation RNA polymerase arabinose araC protein araC–protein complex mRNA Transcription araC gene PBAD rfp gene Plating Tips 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 More Plating Tips 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 Remind Students 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 Sterile technique 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 • DO NOT open plates, observe by viewing through the bottoms •Used plates – dispose in the “contaminated waste” bags P- P+ LB P- P+ LB/amp P+ LB/amp/ara “oops” plates Satellite colonies 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. Why do we get satellite colonies? 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).