Preparation of electroporation competent and cell
transformation
600 μl of overnight cultures were 1:50 diluted into 30 ml fresh LB
medium and grown at 30 °C to an OD600=0.4-0.6. If Red
recombinases harbored by plasmid pSim6 were needed to be expressed,
cultures were then shaken at 42 °C for 15 min in a water bath. Cultures
were centrifugated at 4 °C for 15 min, washed 3~4 times
with 20 ml of cold deionized water. Cell pellet was ultimately suspended
in 0.5 ml of deionized water. 40 μl of fresh prepared competent cells
was used for an electroporation reaction. Electroporation was carried
out using Gene-Pulser (Bio-Rad Laboratories, USA) at 1.8 kv, 25 μF and
200 Ω. Electroporated cells were cultured in 2 ml of LB broth with
shaking for 2 h and then spread onto corresponding counter-selective
agar plates. In detail, for counter-selection marker controlled by
AraC/PBAD inducible system, cells were spread onto LB
agar plates supplemented with 0.4% arabinose and grown at 30 °C; for
counter-selection marker controlled by pL promoter, cells were spread
onto normal LB agar plates at cultivated at 42 °C.