Plasmid construction and molecular biology techniques
The pCGLM1 plasmid we described in (Maroc and Fairhead, 2019) proved
unstable when cloned in E. coli , probably because of the presence
of the f1 ori region, repeated at two different positions in the
plasmid. We decided to construct a new version, pCFYF, shown on Figure
1. We cloned the pMET3 promoter from plasmid pCU-MET3 from
(Zordan et al., 2013), and introduced it in place of the PGK promoter in front of the Cas9 gene in plasmid pJH-2972 from J. E. Haber
(https://protocolexchange.researchsquare.com/article/nprot-5791/v1).
For this, the pMET3 promoter was amplified by PCR with primers
shown in table 2). These primers contain sequences identical to the
borders of the PGK promoter in pJH-2972. Saccharomyces cerevisiae cells from strain W303 (Ralser et al., 2012) were transformed with the
pMET3 promoter PCR fragment along with plasmid pJH-2972,
linearized at the Cla I site, inside the PGK promoter.
Ura+ transformants contained a circular plasmid, where homologous
recombination has resulted in the replacement of the PGK promoter
by the pMET3 promoter. The plasmid was extracted from S.
cerevisiae and transformed into E. coli cells. The new construct
pCFYF (pMET -cas9) was checked by sequencing of the junctions on
both sides of the MET3 promoter by Eurofin
GenomicsTM France.