Extended Data Fig. 3: Impact of TIGER on sensed transcripts.

(a) Recording the essential transcript lpxC in E. coli. (b) Measuring the impact of lpxC Rptr expression in the presence or absence of the base editor. (c) Impact on colony-forming units following transformation of the Rptr plasmid. (d) Impact on culture turbidity after inoculating transformed colonies. (e) Bulk sequencing of lpxC mRNA recording. Colors in b, d and e correspond to the Rptrs shown in a, with orange indicating the scrambled tracrRNA (tracrRNA(scr)). In c - e, lpxC Rptrs were expressed from a high-copy plasmid, with their targets in a low-copy plasmid. (f) Assessing the impact of TIGER on deGFP when recording its expression in E. coli. Two degfp Rptrs were tested in strains with or without the base editor. (g) Impact on deGFP fluorescence during growth in a microplate reader. (h) Impact on degfp transcript levels as determined by RT-qPCR. Two different amplicons covering the Rptr target sites (amplicon 1 for Rptr1, amplicon 2 for Rptr2) within the degfp transcript were selected for relative RNA quantification. Colors in g and h correspond to the Rptrs shown in f, with orange indicating the scrambled tracrRNA (tracrRNA(scr)). (i) Varying the promoter strength driving degfp Rptr expression. (j) Impact on culture turbidity during growth in a microplate reader. (k) Impact on deGFP fluorescence during growth in a microplate reader. (l) Impact on transcript recording via bulk sequencing. Colors in j–l correspond to the promoter strengths in i, while orange represents the scrambled tracrRNA driven by the strongest OR2-OR1 constitutive promoter. In g, h, j, k, and l, the degfp Rptr along with the degfp mRNA were expressed from a low-copy plasmid, while the target was encoded in a medium-copy plasmid. Values in b, d, e, g, h, j, k, and l represent the mean (bar or dot) and standard deviation (error bar or shaded region) of independent experiments starting from three separate colonies.

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