Fig. 2
Schematic description of CRISPRi-mediated repression of endogenous genes involved in byproduct formation. a Genomic structure of four endogenous genes in E. coli. The ldhA and adhE genes are monocistronic, whereas frdA and pta reside in frdABCD and ackA-pta operon, respectively, in E. coli. In the ackA-pta operon, pta is controlled by a separate promoter in addition to the ackA promoter. b The CRISPRi plasmid harboring an sgRNA targeting the pta, frdA, ldhA, or adhE gene in E. coli. c A synthetic fluorescence-based gene-reporter plasmid containing a GFP-encoding gene. Binding sites for each dCas9-sgRNA complex were inserted between the constitutive J23100 promoter (PJ23100) and the gfp gene in the pREGFP3 plasmid. The expression of the gfp gene was controlled by the constitutive J23100 promoter (PJ23100). d, e In vivo fluorescence assay reporting CRISPRi-mediated GFP repression. Two plasmids (pSECRi and pREGFP3) were introduced into E. coli, and expression of the dCas9 protein was induced by 4 mM l-rhamnose. f mRNA levels of CRISPRi target genes in E. coli. The pSECRi plasmid was transformed into E. coli cells, and real-time PCR was performed to determine the mRNA levels of each gene (pta, frdA, ldhA, or adhE). The expression ratios of GFP were calculated as \(expression\, (\% ) = \frac{{{{RFU_{xv} } \mathord{\left/ {\vphantom {{RFU_{xv} } {OD_{xv} }}} \right. \kern-0pt} {OD_{xv} }}}}{{{{RFU_{null} } \mathord{\left/ {\vphantom {{RFU_{null} } {OD_{null} }}} \right. \kern-0pt} {OD_{null} }}}}\; \times \;100,\) where RFU and OD are relative fluorescence units and optical density values at 600 nm, respectively. The subscript xv designates the tested cells harboring the pSECRi plasmid in the presence of l-rhamnose, whereas null indicates a control with the same pSECRi plasmid in the absence of l-rhamnose