Plasmids were linearized with SacI to target its integration into the aox1 gene locus of P. pastoris. The integration of the linearized plasmids was verified by colony PCR and agarose gel electrophoresis using a 0.8% agarose gel, Gel Loading Dye (6X NEB) and 2-Log DNA Ladder (NEB). The transformation of the expression cassette into P. pastoris X-33 was performed according to EasySelect™ Pichia expression guidelines. Briefly, transformations were performed by electroporation using 50 μL of competent cells in 0.1 cm electroporation cuvettes with 3.5 μg of linearized plasmid. Electroporation was achieved with a voltage of 1.5kV, 125 ohms of resistance and a pulse length of 3 ms using a Micropulser system (BioRad, Hercules, CA, USA). Subsequently, cells were transferred in 800 μL YPD supplemented with sorbitol (20% w/v peptone from casein, 10% w/v yeast extract, 4% w/v glucose, 2% w/v agar and 1M sorbitol) at 30°C for 2 h.
Micropulser system
Manufactured by Bio-Rad
Sourced in United States
The Micropulser system is a compact and versatile instrument designed for electroporation, a widely used technique in molecular biology and biotechnology. The system generates precisely controlled electrical pulses that facilitate the introduction of foreign molecules, such as DNA, RNA, or proteins, into a variety of cell types. The Micropulser system is a reliable tool for researchers and scientists working in diverse fields, providing a standardized and efficient approach to cell transformation and transfection experiments.
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Lab products found in correlation
2 protocols using micropulser system
1
P. pastoris Transformation Optimization
2
Electroporation of Phage-Resistant Bacteria
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Phage-resistant isolates were rendered electrocompetent according to the Bio-Rad MicroPulser electroporation apparatus operating instructions and applications guide, section 5 (165-2100). After repetitive washes, a 50-μl cell suspension was mixed with 1 μg plasmid DNA and loaded into a 1-mm electroporation cuvette. Cells were subjected to a single 1.8-kV pulse shock in a Bio-Rad MicroPulser system and then immediately transferred to prewarmed SOC medium and incubated for 1 h before plating on LB agar with 175 μg/ml chloramphenicol. Expression of the wild-type gene insert was induced by exposure to 2% arabinose, followed by incubation for 1 to 12 h before phage challenge. To control for the metabolic effects of arabinose, bacterial isolates transformed with empty vector and exposed to arabinose were tested in parallel.
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