Lab 5a Transformation of Escherichia coli with pARA-R

The “acid test” – does it work? “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.

Differential gene expression 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.

Why do we need arabinose? 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)

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

RFP expression araC gene PBAD rfp gene Transcription mRNA Translation Rfp gene is in place of the araA, araB and araD genes which produce enzymes that digest arabinose in the normal arabinose operon. Translation araC protein

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

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

arabinose – araC protein RFP expression RFP (red fluorescent protein) 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 mRNA Transcription Arabinose is the inducer araC gene rfp gene PBAD

What do we know? 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?

How is Lab 5a different from Lab 5? “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)

Agar Plate tips to tell the students: 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 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

Three Important aspects to stress with your 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 tips for students 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.

Lab 5 day 2 Used plates – dispose in the “contaminated waste” bags 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

“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).

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