Cell-based Assays to Monitor AID Activity

Part I. Measuring mutagenic activity of AID in E. coli
The following procedure has been adapted from Petersen-Mahrt et al. (2002). It provides a measurement of the capacity of AID to mutate the Escherichia coli genome by selecting for those mutations in the rpoB gene that confer resistance to Rifampicin. It can be used as a proxy for the catalytic activity of the enzyme, although it involves deamination of a transcribed gene, which might influence the results. When comparing different AID variants or mutants, it provides a measurement of their mutagenic activity compared to the wt enzyme, which often but not always correlates with the enzymatic activity of the corresponding recombinant enzymes measured on DNA oligonucleotide substrates.

Materials and Reagents

1. Sterile multi-channel basin
   
2. Competent Δung E coli. We use the BW310 strain (Duncan, 1985) (a gift from Dr. Bernard Weiss Department of Pathology, Emory University School of Medicine, Atlanta)
   
3. Plasmids containing AID or AID variants cloned in an inducible prokaryotic expression vector. We use IPTG-inducible pTrcHis vectors (Life Technologies, catalog number: V360-20 )
   Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: V360-20”.
   
4. Tryptone (BioShop Canada, catalog number: TRP402 )
   
5. Yeast extract (BioShop Canada, catalog number: YEX401 )
   
6. Sodium Chloride (NaCl) (BioShop Canada, catalog number: SOD002 )
   
7. Agar (bacteriological grade) (BioShop Canada, catalog number: AGR001 )
   
8. Na₂HPO₄^.7H₂O (BioShop Canada, catalog number: SMP400 )
   
9. Potassium Phosphate Monobasic (KH₂PO₄) (BioShop Canadap, catalog number: PPM666 )
   
10. Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: A0171 )
    
11. Ampicillin (stock 100 mg/ml in ddH₂O) (BioShop Canada, catalog number: AMP201 )
    
12. Carbenicillin (100 mg/ml in ddH₂O) (BioShop Canada, catalog number: CAR666 )
    
13. Rifampicin (stock 100 mg/ml in DMSO) (Sigma-Aldrich, catalog number: R3501 )
    
14. IPTG (stock 1 M in ddH₂O) (BioShop Canada, catalog number: IPT001 )
    
15. Transparent sterile 96-well plates, flat bottom (Corning, catalog number: 3595 )
    
16. LB media (see Recipes)
    
17. LB agar (see Recipes)
    
18. 2x TY media (see Recipes)
    
19. 5x M9 media (see Recipes)
    

Equipment

1. Bunsen burner
   
2. Sterile glass beads (for bacteria plating) (Genlantis, EZ-spread, catalog number: C400050 )
   
3. 37 °C incubator (for bacterial plates)
   
4. 37 °C incubator with shaker (for bacterial cultures)
   
5. 42 °C water bath
   
6. Hand counter
   
7. Multichannel pipettor
   

Procedure