Conjugation of the TransBac library plasmids
Hfr strain is the donor strain that carries each TranBac library
plasmid and can transfer the target plasmid by conjugation (unpublished
resource by Mori). The donor strain was grown on LB agar plates
supplemented with tetracycline (Tc) and 25-50 µg/mL diaminopimelic acid
(DAP). Hfr strain requires DAP because of the deletion of thedapA gene. Well-grown donor and acceptor cell cultures were mixed
1:1 ratio in a 1.5 mL polypropylene tube and incubated at 37°C for 1
hour without shaking. After the appropriate time for conjugation, the
cell mix was plated onto LB agar plates containing Tc (12.5 µg/mL)
without DAP. The plates were incubated at 37°C overnight.