Figure 2.

Functional complementation of E. coli polA12^ts by (a) wild-type E. coli pol I (positive control), (b) Tt72 pol, (c) T. thermophilus MAT72 pol I, (d) T. aquaticus pol I, and (e) vector pHSG576 (negative control). E. coli polA12^ts JS200 [pBR322] was transformed with pHSG-Pol-I-wt (Eco pol I), pHSG-polTt72 (Tt72 pol), pHSG-polTth (Tth pol), pHSG-polTaq (Taq pol), and plasmid pHSG576 (low-copy plasmid with a pol I-independent pSC101 origin replication). Transformed cells were grown to A600 of 0.5 in LB medium containing tetracycline (15 µg/mL), chloramphenicol (34 µg/mL), and ampicillin (100 µg/mL). Then, the cells were serially 10-fold diluted with LB medium, and 5 µL of each dilution was spotted on LA plates supplemented with the same antibiotics and IPTG at a concentration of 1 mM. Plates in duplicate were incubated at 30 °C and 42 °C for 24–30 h.