Gene pulser

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.

Candidate genes were amplified from wt S. marcescens VA using primers indicated Supplementary Table 2 and cloned into pBBR1MCS-2 (kind gift from Kenneth Peterson – Addgene plasmid #85168; Kovach et al., 1995 (link)) in E. coli. Plasmid sequences were verified by Sanger sequencing. Each recombinant plasmid was then electroporated with a GenePulser (Bio-Rad) in electrocompetent S. marcescens C3 cells, prepared with the GenePulser according to the manufacturer’s instructions. For each gene, bacteria were plated on LB supplemented with kanamycin (50 μg/mL) to select those carrying the plasmid.

Electro-competent cells were prepared following a standard protocol [35 (link)]. Plasmid DNA or Gibson assembly reactions (~100 ng) was used to transform of E. coli competent cells (~10⁹ cells in 50 μl 10% v/v glycerol in water). Electroporation of E. coli was performed with a Bio-Rad gene pulser with a setting of 200 Ω, 25 μF, and 1.75 kV in a 0.1 cm cuvette. Immediately after electroporation, SOC medium (1 mL) was added and cells were incubated (37°C for 1 h); the recovered cells (100 μL)were then spread on LB agar containing appropriate antibiotic for selection and the plates were incubated (37°C overnight).
For Z. mobilis transformation, plasmid DNA (~1 μg) was used to transform cells (~10⁹ cells in 50 μl 10% v/v glycerol in water). Type 1 restriction inhibitor (1 μL; Epicentre) was added to the plasmid DNA prior to mixing with competent cells. Electroporation of Z. mobilis was performed with a Bio-Rad gene pulser with a setting of 200 Ω, 25 μF, and 1.6 kV in a 0.1 cm cuvette. Immediately after electroporation, recovery broth (1 mL; 5 g glucose/L, 10 g yeast extract/L, 5 g tryptone/L, 2.5 g (NH₄)₂SO₄/L, 0.2 g KH₂PO₄/L, and 0.25 g MgSO₄•7H₂O/L) was added and the cells were incubated (2–3 h at 30°C). The recovered cells were spread on RMG-agar containing the appropriate antibiotic for selection and the plates were incubated (30°C for 2–4 d) to obtain transformed colonies.