Electrotransformation optimization of plasmid pGAPZαA – CecMd3cs into Pichia pastoris GS115 with response surface methodology
Abstract
Background: Industrial production is increasingly reliant upon recombinant microorganisms across sectors. Therefore, it is of great importance for long-term operational viability that care be taken in optimizing the preparation of operationally key organisms. In electrotransformations, low-frequency electric field pulses increase yeast cell viability but decreases their transmembrane of recombinant DNA vectors. Herein, we investigated the optimal parameters for transforming pGAPZαA-CecMd3cs into Pichia pastoris GS115 and obtaining the efficient production of recombinant cells of interest.
Results: For an initial experiment, we utilized a single-factor experimental design to perform the electroporation and measure the vector transformation efficiency in triplicate, identified three out of five voltages (250–350 V), pulse durations (9–17 ms), and pulse frequencies (1–5 pulses) as candidate values for further analysis. Based on this data, we obtained the optimal value for each parameter by utilizing Box–Behnken of response surface methodology statistical modelling. We found that under a fixed voltage, the primary factors affecting electrotransformation efficiency were pulse duration and frequency. Furthermore, we identified synergistic interactions between pulse length and voltage and between pulse length and frequency.
Conclusions: The optimal parameters for electroporation are 275 V and 3 pulses of a 15-ms duration. We bioinformatically determined the optimal transformation parameters.