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.