YTK12 Chemically Competent Cell

YTK12 Chemically Competent Cell

The YTK12 strain is a sucrose invertase (SUC2) deficient type of Saccharomyces cerevisiae. The yeast sucrose invertase (SUC2) can hydrolyze sucrose or raffinose into glucose and fructose; it participates in carbohydrate catabolism; it can localize in the cytoplasm and be secreted to function in two different forms. The N-terminus of this gene has a secretion signal peptide, which directs the secretion of sucrose invertase to the extracellular space. The SUC2 gene in the YTK12 strain is mutated and cannot synthesize sucrose invertase. A functional sucrose invertase gene is introduced through the yeast plasmid pSUC2, but the N-terminal signal peptide of the pSUC2 empty vector is removed, making it unable to be secreted outside the cell. By linking different peptides to the N-terminus of the SUC2 protein, the secretion functionality of the linked peptides can be verified. The YTK12 yeast strain is a SUC2 gene deficient strain and cannot grow normally on YPRAA medium with raffinose as the sole carbon source. Only by linking a secretion-active signal peptide sequence to the N-terminus of the SUC2 gene in the pSUC2 vector and transforming it into the YTK12 strain, can the sucrose invertase be properly secreted. The YTK12 yeast can then convert raffinose into glucose and grow normally on YPRAA medium (Jacobs et al 1997, Oh et al 2009). Additionally, the hydrolysis product of sucrose invertase (SUC2), glucose, can reduce 2,3,5-Triphenyltetrazolium chloride (TTC) to an insoluble red substance, 1,3,5-Triphenylformazan (TPF), and the color reaction can also be used to determine if the verified peptide has secretion functionality. The transformation marker for the YTK12 strain is: trp1, his3, ura3; YTK12 competent cells are produced through a special process and can be stored at -80℃ for 12 months, with the pSUC2 plasmid detecting a transformation efficiency of >10⁴ cfu/μg DNA.

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