Influences of plasmid forms and electric pulses on transformation efficiency in yeast using electroporation
Paper Details
Influences of plasmid forms and electric pulses on transformation efficiency in yeast using electroporation
Abstract
The electroporation systems was widely used for transforming eukaryotic gene. The purpose of this research was to compare effects of plasmid forms and electric pulses on transformation efficiency in yeast using various electroporation approaches. The pPICZαA-crypt plasmid, containing the beta-cryptogein gene, was digested and undigested by SacI to produce linearized- and circular-plasmid, respectively. Both plasmid forms were transformed into competent cells of Pichia pastoris (Y11430) using either electric pulseof 1.5 or 2.0 KV. The single colony was selected and cultured in YPD medium, containing 100 µg/ml zeocinTM, and successful transformants were confirmed by using PCR-amplification.The result showed that the linearized plasmid in both electric pulses significantly generated higher transformants (average 29.25-30.00 colonies/plate) than the circular plasmid (7.63-8.13 colonies/plate). Twelve putative transformants in each transformation system were assessed successful transformation efficiency through PCR with present of the inserted crypt gene and AOXI. The result showed that transformation efficiency of the linearized plasmid (12 of 12 transformants) was significantly higher (p=0.01) than the circular plasmid (10 of 12 transformants) in both electric pulses. This study indicated that the linearized plasmid might be used for increasing electroporated transformation efficiency in P. pastoris.
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