Fig. 3.
Overview of predicted and observed integration events in P. pastoris. a The plasmid pAHBgl-GFP is restricted with BglII, excising the expression cassette containing the gfp gene and a selection marker. The AOX1 promoter (pAOX1) and a homology sequence downstream of AOX1 (3′ AOX1 HS) facilitate a replacement of the chromosomal AOX1 locus with the expression cassette via double crossing over (solid crossed lines). After homologous recombination the expression cassette is stably integrated into the genome. b1 Via single cross over the expression cassette is inserted up- or downstream of the AOX1 locus. This event can occur multiple times, leading to tandem integrations. Other additive integrations can also happen multiple times up- or downstream of AOX1. b2 Additive integration of the whole pAHBgl-GFP plasmid at the AOX1 locus with highlighted BglII site. b3 Replacement of a region downstream of AOX1 with only the selection marker. The event is facilitated by a double crossing over event using 3′ AOX1 HS and AOX1 TT (dashed crossed lines in a). c Inversion of the E. coli backbone in comparison to the adjacent expression cassettes in the MutS clone JPS233