Fig. 1: Development of the PI-seq workflow.
A A total of 5541 sequences, each 140 bp sequences immediately upstream of annotated start codons, were computationally extracted from P. simiae WCS417 genome. These promoter sequences were categorized into three groups according to the distances to upstream genes: group I (≤30 bp), group II (31–139 bp), and group III (≥140 bp). B Each promoter library was synthesized, barcoded with 23 bp sequences, and cloned into the CRAGE accessory vector (pW26_mod), which included an apramycin-resistant marker (AprR) flanked by two mutually exclusive lox sites (lox2272 and lox5171), necessary for site-specific recombination. C The plasmids were transformed into an E. coli donor strain and conjugated into P. simiae WCS417 (strain SB599), which harbored a CRAGE landing pad on the chromosome. The landing pad is located between locus tags PS417_RS26490 and PS417_RS26495, without disrupting any coding sequences34. Cre-mediated recombination integrated the barcoded promoters into the landing pad. D The promoter-barcode regions were amplified from the genomic DNA and sequenced via targeted DNA-seq to associate each promoter with its corresponding unique barcodes. E The transcriptional activity of promoters was characterized by growing cells under various conditions, extracting DNA and RNA, and amplifying barcode regions from the DNA and cDNA. The cDNA was synthesized from RNA by selective reverse transcription. Promoter activities were then quantified by normalizing RNA barcode counts to DNA barcode counts for each gene.
