Fig. 1
From: Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
Design of Cas12a-dependent mutagenesis of the fimAICDFGH operon. A Scheme of the fimAICDFGH operon (i). Its transcription depends on a promoter (fimAp) that is part of an invertible DNA element indicated by a white dotted line upstream of fimA (ii). The target sites for Cas12a crRNA binding sites in fimA are shown as horizontal lines (ii). The site of the 97 bp deletion at position 75 of the fimA gene is illustrated as a gap (iii). Positions 1 and 75 in fimA where the L3S2P56 terminator sequence was inserted to generate mutants ΔfimA1::L3S2P56 and ΔfimA75::L3S2P56 are marked on iii. The lines converging on an arrow denote the regions spanning the L3S2P56 terminator sequence insertions (iii). The ribonucleotide sequence and expected folding of the RNA corresponding to the transcriptional terminator L3S2P56 (iv). B Analysis of fimA mutants by agarose gel electrophoresis of PCR products. Lanes 1, 3, and 5, show a 514 bp PCR product generated by primers F1 and R1 (shown in ii) that amplify a fragment of fimA from wild type E. coli. Lane 2 shows the unchanged migration pattern of the PCR product compared to WT using primers F1 and R1 on template DNA from the stop codon mutant (fimAA106T). Lanes 4 and 6 show the PCR product generated using primers F1 and R1 after amplifying template DNA from the deletion mutant (ΔfimA75, 420 bp) and the internal terminator insertion (ΔfimA75::L3S2P56, 474 bp), respectively. Lane 7 shows a 988 bp PCR product generated by primers F2 and R2 (shown in iii) that amplify a fragment of fimA from wild type E. coli including the leader sequence and lane 8 shows the 870 bp PCR product generated with primers F2 and R2 on template DNA from the second terminator insertion (ΔfimA1::L3S2P56). Lane L was loaded with a double-stranded DNA ladder