Kanamycin as a selectable marker for Pichia pastoris

Poster Number

3

Format

Poster Presentation

Abstract/Artist Statement

The yeast Pichia pastoris is commonly used as a host organism in heterologous protein expression. One of the main problems of P. pastoris is that it does not have a wide range of selectable markers available for use in transformation processes. We adapted a kanamycin resistance selectable marker to fulfill the same function of the zeocin resistance selectable marker. With the use of Real-Time PCR, we have quantified the varying amounts of the cassette transformed in the yeast colonies. This data was consistent with the Southern Blot data from preliminary experiments which supported the fact that the sizes of the yeast colonies were roughly proportional to the number of cassettes that were transformed into their genome. In short, we have been able to produce a plasmid that contains the kanamycin selectable marker and a beta-lactamase reporter gene. Through the use of an activity assay we found that the larger colonies with a higher cassette count within their genome produced more beta-lactamase. The Southern Blot, activity assay and Real-Time PCR findings all have supported the hypothesis that the copy number of the yeast transformation can be linked to the size of the colonies, with the larger colonies having a higher copy number of the kanamycin selectable marker.

Location

Pacific Geosciences Center

Start Date

30-4-2005 1:00 PM

End Date

30-4-2005 3:00 PM

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Apr 30th, 1:00 PM Apr 30th, 3:00 PM

Kanamycin as a selectable marker for Pichia pastoris

Pacific Geosciences Center

The yeast Pichia pastoris is commonly used as a host organism in heterologous protein expression. One of the main problems of P. pastoris is that it does not have a wide range of selectable markers available for use in transformation processes. We adapted a kanamycin resistance selectable marker to fulfill the same function of the zeocin resistance selectable marker. With the use of Real-Time PCR, we have quantified the varying amounts of the cassette transformed in the yeast colonies. This data was consistent with the Southern Blot data from preliminary experiments which supported the fact that the sizes of the yeast colonies were roughly proportional to the number of cassettes that were transformed into their genome. In short, we have been able to produce a plasmid that contains the kanamycin selectable marker and a beta-lactamase reporter gene. Through the use of an activity assay we found that the larger colonies with a higher cassette count within their genome produced more beta-lactamase. The Southern Blot, activity assay and Real-Time PCR findings all have supported the hypothesis that the copy number of the yeast transformation can be linked to the size of the colonies, with the larger colonies having a higher copy number of the kanamycin selectable marker.