Fig. 3

Production and testing of precursor plasmid v1 and nChr 1. (a) Incorporation of an initial, one-off, gene-landing pad into the precursor plasmid. Work on eDA83 was halted due to leaky I-SceI expression in E. coli. (b) Following deletion of I-SceI in eDA83 by insertion of Kan^R (creating eDA110), Tel was inserted to yield eDA137, the precursor plasmid (v1) of nChr 1. The plasmid was linearized in vitro and used to transform (“L&T” in figure) CBS7435 K. phaffii cells creating strain yDA122. The agarose gel shows I-SceI digestion of Tel-containing eDA137 (versus control, i.e. no-Tel, eDA110). (c)  Despite nChr 1 appearing stable in chromosome-loss assays performed on yDA122, WGS (and subsequently, colony PCR, see gel) revealed major chromosomal rearrangements as indicated in the schematic. Note, in yDA122, the loss of PCR-product 3 (of nChr 1), but retention of PCR-product 2 of the suspected translocation product nChr 1* (i.e. nChr 1(p):Chr 3(q))