Figure 3

Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the cultures to a final concentration of 1 mM; the fifth culture received no IPTG. The cells were heat shocked for the final 15 minutes, prepared for electroporation and electroporated with DNA (~0.25 μg) containing the kan gene flanked by 40 bp of EHEC DNA (resulting in a deletion of O-islands #130 and #131). After suspension in LB, the cells were grown for 90 minutes at 37°C and plated on LB plates containing 20 μg/ml kanamycin. The number of Kan^R transformants per 10⁸ survivor and total number of Kan^R transformants are plotted as a function of IPTG concentration. The data points are averages of two experiments (ranges are shown). A random check of 160 colonies showed that 95% re-struck successfully to fresh LB plates containing 20 μg/ml kanamycin; 10 of 10 of these colonies were verified by PCR analysis to be true recombinants (data not shown). Insert: 0.1 ml of electrocompetent cells, prepared with and without 1 hour IPTG induction, were spread on LB plates containing 100 μg/ml rifampicin to determine total number of Rif^R mutants. Dilutions of the cells were titered on LB plates to determine total cell density; experiments done in triplicate (+/- standard error).