Fig. 2

CRISPR-dCas9 setup in JE1-producing strains. A Schematic overview of chromosome integrated constructs in strains used for CRISPRi. je1zyn is a synthetic gene encoding an α-amylase placed upstream to chloramphenicol acetyltransferase (cat) that allow chloramphenicol resistance selection. Dead Cas9 (dCas9) binds specific DNA regions based on sequence complementarity of the single guide RNA (sgRNA) to block transcription at said region. Spectinomycin selection is allowed by insertion of a specR gene. Both dCas9 and JE1zyn are expressed from a strong promoter (P4199) [15]. je1zyn and dCas9 are inserted in the pel and amyE loci, respectively. Single guide RNAs are expressed from a Pq promoter and inserted in the alr locus. B Biolector fermentation JE1 activity of strains expressing JE1zyn and JE1zyn + dCas9 relative to JE1zyn. N = 3, error bars depict standard deviation. C qRT-PCR je1zyn mRNA levels of strains expressing JE1zyn and JE1zyn + dCas9 relative to JE1zyn. N = 3, error bars depict standard error of the mean. (D) as (B) but strains expressing JE1zyn + dCas9 + sgRNA::gfp (sgRNA::GFP) and JE1zyn + dCas9 + sgRNA::JE1zyn (sgRNA::JE1zyn). (E) as (C) but strains expressing JE1zyn + dCas9 + sgRNA::gfp (sgRNA::GFP) and JE1zyn + dCas9 + sgRNA::je1zyn (sgRNA::JE1zyn)