Fig. 2
Optimization of electrotransformation procedure in Acetobacterium wieringae strain JM using the plasmid pMTL83151. a Effect of harvesting OD600 during the preparation of A. wieringae JM competent cells on transformation efficiency, ET. Growing cells were sampled during exponential phase (OD600 0.21, 0.36, 0.43, 0.51, 0.60, 0.86) for preparation of competent cells prior electroporation. After resuspension of competent cells in SMP 10% DMF, all conditions had an OD600 of 30. b Effect of the number of washing steps during preparation of A. wieringae JM competent cells on transformation efficiency. Cells were grown to OD600 of 0.36 and were sampled to be washed 1, 2, 3, 4, and 5 times with SMP buffer during the preparation of competent cells before electroporation. c Effect of pH of resuspension buffer on transformation efficiency. Cells were grown to OD600 of 0.40, washed in SMP buffer pH 6, and were sampled to be resuspended in SMP 10% DMF pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 during competent cell preparation before electroporation. d Effect of pulse voltage (field strength) on transformation efficiency. ET was measured using electric pulses of 1.0, 1.5, 1.8, 2.0, and 2.5 kV, corresponding to field strengths of 5.0, 7.5, 8.0, 10, and 12.5 kV/cm. e Effect of plasmid DNA amount on colony-forming units (CFU) and transformation efficiency. Separately, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 μg of plasmid corresponding, respectively, to cell–plasmid ratios of 12.0, 6.0, 3.0, 2.0, 1.5, and 1.2, were added to competent cells of A. wieringae JM and electroporated. The total number of colony-forming units and transformation efficiency was quantified. f Effect of post-electroporation incubation time on transformation efficiency. Cells were electroporated, transferred to a 3 mL outgrowth medium, and incubated for 2, 4, 6, 8, and 20 h before selective plating