Escherichia coli transformation was performed by chemical transformation. Transformation of L. plantarum by electroporation was performed as previous described [38 (link)]. Briefly, L. plantarum cells cultured to mid-exponential phase (OD600 ~ 0.4–0.6) were collected, washed twice with SM buffer (952 mM sucrose supplemented with 3.5 mM MgCl2) and resuspended in SM buffer. Plasmid DNA ( ~ 1 μg) was added to 100 μL prepared competent cells. The resulting cell mixture were incubated on ice for 10 min. Cell mixture were electroporated using 2 mm electroporation cuvette and Gene Pulser (BioRad) under following condition: 2000 V, 25 μF, 400 Ω. Cells was recovered in SMRS broth (MRS broth supplemented with 0.5 M sucrose and 0.1 M MgCl2) at 37 °C for 3 h before spreading on MRS agar plates containing erythromycin.
Transformation of P. putida by electroporation following previously published protocol [39 (link)]. P. putida cells cultured to mid- exponential phase (OD600 ~ 0.2–0.4) were collected, washed twice with 300 mM sucrose solution and resuspended in 300 mM sucrose. After 10 min incubation on ice, the mixture of competent cells and DNA ( ~ 1 μg) was electroporated using 2 mm electroporation cuvette and Gene Pulser (BioRad) under following condition: 2500 V, 25 μF, 400 Ω. Cells was recovered in LB broth at 30 °C for 2 h before spreading on LB agar plates containing kanamycin.
Transformation of P. putida by electroporation following previously published protocol [39 (link)]. P. putida cells cultured to mid- exponential phase (OD600 ~ 0.2–0.4) were collected, washed twice with 300 mM sucrose solution and resuspended in 300 mM sucrose. After 10 min incubation on ice, the mixture of competent cells and DNA ( ~ 1 μg) was electroporated using 2 mm electroporation cuvette and Gene Pulser (BioRad) under following condition: 2500 V, 25 μF, 400 Ω. Cells was recovered in LB broth at 30 °C for 2 h before spreading on LB agar plates containing kanamycin.
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