0.1 cm electroporation cuvette

Electroporation was used for plasmid transformation as described previously^{41 (link)} with modifications. After overnight cultivation in 5 mL LB medium at 30 °C and 250 rpm, 1 mL P. putida cells were centrifuged and washed twice with 1 mL of 0.3 M sucrose solution. Then, cell pellets were resuspended in 0.2 mL of 0.3 M sucrose solution. Plasmid DNAs (0.2–2 μg) were mixed with 100 μL cell suspension in 0.1 cm electroporation cuvette (Bio-Rad) and electroporated at 1.6 kV by Eporator (Eppendorf, Germany). Subsequently, cells were transferred to 1 mL LB medium for recovery at 30 °C and 250 rpm for 1–2 h. 100 μL recovered cells were plated on LB agar plates containing appropriate antibiotics and incubated at 30 °C overnight. Colonies were picked up from agar plates to selective LB medium and grown at 30 °C and 250 rpm overnight for further verification by target fragment PCR and plasmid digestion. For sucrose counter-selection, colonies selected on LB plates were incubated at 30 °C and 250 rpm for 4–6 h. Then, 100 μL cells were plated on LB + 10% sucrose agar plates overnight. Finally, 20–40 colonies were selected to screen for correct gene replacements or knock-out by PCR.

Either purified plasmid DNA, ligation or recombination reactions was added to the electrocompetent bacteria and carefully mixed. Following a 1-min incubation on ice, the suspension was transferred to a pre-chilled 0.1 cm electroporation cuvette (Bio-Rad, cat. #1652089) and electroporated using Ec1 programme on MicroPulser Electroporator (Bio-Rad, cat. #1652100). Following this, 1 ml SOC medium was added to the cuvette and transferred to a microcentrifuge tube. For recovery, bacteria were incubated at 37 °C in an incubator shaker (Infors, HT Multitron, cat. #2292113-18) at 225 rpm for 1 h. Following collection using centrifugation at 400–600 × g, bacteria were resuspended in LB medium and serial dilutions were plated onto LB agar plates containing 50 mg/ml ampicillin and were incubated at 30 or 37 °C overnight depending on the bacterial strain used.

Electroporation of E. coli was carried out according to the procedures as follows. Fresh colony was picked from LB agar plates, inoculated into a tube containing 1 mL LB medium and grown overnight at 37 °C with shaking at 250 rpm. This seed culture was used to inoculate a 250-mL flask containing 50 mL LB medium at 37 °C aerobically with shaking at 250 rpm to increase cell mass. Cells at exponential phase were collected and incubated in ice for 30 min. Cells were washed and suspended in chilled 10% glycerol, and 50 μL aliquots of cells were transferred to a 1.5-mL centrifuge tubes. 1 μg of DNA sample was mixed with cells and incubated on ice for 5 min before the mixture was transferred to a chilled 0.1 cm electroporation cuvette (Bio-Rad, Hercules, CA). An electric pulse (18 kV/cm,25 μF capacitance and 200 Ω resistance) was applied to the cuvette for electroporation. Then cells were suspended in 1 mL LB and incubated in a tube at 37 °C for 40 min before plated on LB agar plates containing appropriate antibiotic. The plates were incubated at 37 °C overnight then.