Journal: Microbial Cell Factories

Article Title: A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material

doi: 10.1186/s12934-023-02262-4

Figure Lengend Snippet: 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 ( v 1) 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-Sce I 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))

Article Snippet: To achieve this, the 13.5-kb Sph I-digested and gel-extracted eDA83 was ligated with a 980-bp PCR-amplified Kan R (using oligos 321/322 and vector pUC57-Kan (Addgene) as a template).

Techniques: Plasmid Preparation, Expressing, In Vitro, Agarose Gel Electrophoresis, Translocation Assay