Conjugation of the TransBac library plasmids
Hfr+ strain (E. coli F+dapA- strain; BW38029 Hfr (CIP8oriT::cat ) dap- ) is the donor strain
that carries each TransBac library plasmid and can transfer the target
plasmid by conjugation (Otsuka et al. , 2015; Bobonis et
al. , 2022). The donor strain was grown on LB agar plates supplemented
with 12.5 µg/mL tetracycline (Tc) and 25-50 µg/mL diaminopimelic acid
(Acros Organics, #235540050). Hfr+ donor
strains require DAP to grow because of the deletion of the dapA .
Well-grown donor and acceptor cells were mixed (1:1) in a 1.5 mL
polypropylene tube and incubated at 37°C for 1 hour without shaking. The
recovery step was carried out in SOC medium for 1 hour at 37°C 850 rpm
shaking thermostat (Eppendorf Thermomixer compact), and the cells were
spread onto LB agar plates containing only Tc without DAP. The plates
were incubated at 37°C overnight. The ORFs were sequenced using ISM43
(TTA AAG AGG AGA AAC CTA GG) and ISM44 (CTT AGC GGC CGC ATA).