Fig. 6

Design of Cas12a-dependent mutagenesis of the atpIBEFHAGDC operon. A Scheme of the atpIBEFHAGDC operon (i). The 816 bp deletion of the atpB gene is illustrated as a gap and the lines converging on a vertical arrow denote the L3S3P41 insertion at that position to generate mutant atpB::L3S3P41 (ii). Included here is also the ribonucleotide sequence and expected folding of the RNA corresponding to the L3S3P41 transcriptional terminator (ii). The insertion of the L3S2P56 terminator sequence upstream of atpI and downstream of the atpIp promoter that generated the atpIp::L3S2P56 mutant is illustrated as a dotted arrow (iii). The target sites for Cas12a crRNA binding sites in atpIp and atpB are shown as horizontal lines (iii). Horizontal arrows indicate positions corresponding to DNA primers used in PCR analyses, here named F3, R3, R4 and R5. (i, ii, iii). B Analysis of atp mutants by agarose gel electrophoresis of PCR products. Lane 1 shows the 1837 bp PCR product generated by primers F3 and R4 that amplify a segment of the genes atpIB in genomic DNA from wild type E. coli. Lane 2 shows the migration pattern of the 1021 bp and 1068 bp PCR products on DNA from the E. coli atpB and atpB::L3S3P41 mutants generated by primers F3 and R4, respectively. Lanes 4 and 5 show a 521 and 575 bp PCR products generated by primers F3 and R5 that amplify a segment of the promoter atpIp and the atpI gene on DNA from the WT E. coli and atpIp::L3S2P56 mutants, respectively. Lane 7 shows a PCR fragment produced by primers F3 and R3 that amplify a 385 bp product present only in the E. coli ΔatpIBEFHAGDC mutant but not in the WT strain (lane 6). Lane L was loaded with a double-stranded DNA ladder