Pulse controller

The recombinant plasmids pLN‐hsCD80, pLN‐CTB‐hsCD80, or empty vector pLN were transformed into L. lactis NZ3900 by electroporation using a Gene Pulser and Pulse Controller apparatus (Bio‐Rad, USA) at 2.5 kV, 25 μF, and 200 Ω as described previously.³⁰ The transformed L. lactis were selected using Elliker medium with lactose as the sole carbon source. Positive colonies were identified by PCR, restriction enzyme digestion, and DNA sequencing. The recombinant L. lactis transformed with pLN, pLN‐hsCD80, or pLN‐CTB‐hsCD80 was subsequently referred to as L‐vector, L‐hsCD80, and L‐CTB‐hsCD80, respectively.

1.5 × 10⁶ U373 cells were transfected with specific STAT3 siRNA transfection (Santa Cruz Biotechnology, sc-29493) and control siRNA-A (Santa Cruz Biotechnology, sc-37007) as a scrambled control for STAT3 knockdown, or with sip53 plasmid and empty vector as control for p53 knockdown, by electroporation using the Bio-Rad Pulse Controller at 180 V, according to the manufacturer's instructions, and cultured in 24 well plates. After 48 h cells were lysate and protein extracts were subjected to western blot analysis.

Heat‐shock transformation of E. coli was performed as described previously (Green and Sambrook, 2018 ). Transformed cells were selected in LB plates supplemented with ampicillin (100 µg ml⁻¹) or streptomycin (60 µg ml⁻¹). P. putida KT2440 and GN442ΔPP_2426 electrocompetent cells were prepared as described previously (Martínez‐García and de Lorenzo, 2012) with slight modifications. Overnight cultures were transferred to a fresh LB medium with the OD₆₂₀ adjusted to 0.1 and then cultivated in shake flasks until OD₆₂₀ reached 0.8. Cells in mid‐exponential‐phase were washed two times with sucrose (300 mM) and re‐suspended in the same solution. Freshly prepared electrocompetent cells and plasmids (100 ng) were mixed in an electroporation cuvette with 2 mm gap width. The electric pulse was applied using a Gene Pulser apparatus equipped with a Pulse Controller (Bio‐Rad, Hercules, CA, USA). Subsequently, cells were plated on M9 plates with citrate (10 mM) or vanillylamine (5 mM) as sole carbon source and streptomycin (100 μg/ml) for the selection. For the growth‐based ATA selection method, 1 mM IPTG was added to induce gene expression. Cells were diluted 1000x and 10 000x using a sodium chloride solution (0.9% NaCl) prior to plating. Plates were incubated at 30 °C for 3 days.

1.5 × 10⁶ U373 cells were transfected with empty vector or sip53 plasmid for p53 knockdown by electroporation using the Bio-Rad Pulse Controller at 180 Volts, according to the manufacturer’s instructions, and cultured in 24-well plates. After 24 h, cells were treated or not with PBA (7.5 mM) for an additional 24 h. After that period, a trypan blue assay was performed, cells were lysated and protein extracts were subjected to Western blot analysis.