Electrotransformation optimization of plasmid pGAPZαA - CecMd3cs into Pichia pastoris GS115 with response surface methodology

Electronic Journal of Biotechnology

Available online 25 November 2022

Electronic Journal of BiotechnologyAuthor links open overlay panelAbstractBackground

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.

Keywords

Electric field

Electroporation

Electrotransformation

Parameters

pGAPZαA-CecMd3cs

Pichia pastoris GS115

Plasmids

Yeast

AbbreviationsRSM

response surface methodology

RT-PCR

reverse transcription-polymerase chain reaction

YPD

Yeast Extract Peptone Dextrose Medium

© 2022 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V.

留言 (0)

沒有登入
gif