Megax dh10b t1

E. coli chemically competent cells were either DH5α (NEB) or TOP10 (Invitrogen). High-competent electrochemical competent cells MegaX DH10B T1 (Invitrogen) were used for the random library plasmid transformation. M. pneumoniae M129 (passage 34) was grown in modified Hayflick medium in 150 cm² flasks and transformed by electroporation as previously described^{46 (link)}. Briefly, cells were split 1:10, and washed and collected in 300 µl Electroporation buffer (8 mM HEPES·HCl, 272 mM sucrose, pH 7.4) 3 days later. Fifty-microliter cells were used to electroporate 5 µg plasmid (1 mm gapped cuvettes, 1.25 kV, 100 Ω, 25 µF, in a Gene Pulser Xcel Electroporator, Bio-Rad). Cells were recovered in Hayflick at 37 °C for 2 h and diluted 1:5 in Hayflick supplemented with 80 µg ml⁻¹ Gentamycin.

Using a single colony or a frozen glycerol stock, a liquid culture of the bacterial strain One Shot ccdB Survival 2 T1 (Invitrogen, cat. #A10460) or MegaX DH10B T1 (Invitrogen, cat. #C640003) was set up in 100 ml antibiotic-free LB medium and incubated at 37 °C in an incubator shaker (Infors, HT Multitron, cat. #2292113-18) at 200 rpm overnight. The following day, 10 ml of the overnight culture was added to 200 ml pre-warmed LB medium and grown at 37 °C in an incubator shaker (Infors, HT Multitron, cat. #2292113-18) at 200 rpm until an OD₆₀₀ = 0.5 was reached. The bacterial suspension was chilled on ice for 20 min and centrifuged at 4 °C at 2200 × g for 15 min. The supernatant was discarded, and the bacterial pellet was resuspended in 200 ml ice-cold 10% glycerol. After centrifugation, the supernatant was discarded, and the bacterial pellet was resuspended in 50 ml ice-cold 10% glycerol. The bacterial suspension was spun-down, the supernatant was discarded, and the pellet was resuspended in 8 ml ice-cold 10% glycerol. After final centrifugation, the bacterial pellet was resuspended in 600 μl ice-cold 10% glycerol and directly used for electroporation or stored at −80 °C.