New heterologous modules for classical or PCR‐based gene disruptions in <i>Saccharomyces cerevisiae</i>

TLDR

The authors present a dual‑module system that permits a single transformation to generate in‑frame gene substitutions, enabling simultaneous promoter activity testing and phenotype screening. They engineered a kanMX resistance cassette fused to Ashbya gossypii TEF regulatory elements and a lacZMT reporter cassette, both inserted into a new multiple‑cloning sequence with 18 unique sites and flanked by direct repeats for excision, facilitating efficient selection and PCR‑based gene disruption. The kanMX module conferred robust G418 resistance and, in PCR‑based disruptions with only 35‑base homology arms, all 12 tested colonies carried the correctly integrated cassette.

Abstract

Abstract We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading‐frame of the E. coli transposon Tn 903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading‐frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3′ end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in‐frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10–3–10–4. The 1·4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR‐added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin‐resistant colonies carried the correctly integrated kanMX module.