Fig. 1: An RNA-Seq approach for high-throughput aTF characterization.

a The space of allosteric transcription factors (aTFs) and small molecules that can be sensed with different approaches. Black points represent specific ligand:aTF pairs. The dark green circle represents the extent to which existing methods can currently expand aTF:ligand affinity. The light green circle represents the extent to which new methodologies must increase aTF:ligand pairs. b Construct design pairs aTF variant activity to transcription of randomized barcodes. c Construction and mapping of the aTF library to barcodes. d Workflow to acquire ligand specificity profiles for each aTF variant across different ligands. e Box plot showing distribution of F-scores for barcodes mapped to each TtgR variant via RNA sequencing (RNA-Seq) after induction with naringenin. The number of barcodes for each variant is described in Supplementary Fig. 1. The box represents the interquartile range (IQR). Whiskers extend to 1.5 times the IQR. Fliers denote points that lie outside the whiskers. Center line represents median. Filled blue squares indicate the presence of the corresponding mutation in the variant. f Correlation of quantitative reverse transcription polymerase chain reaction (qRT-PCR) fold enrichment and RNA-Seq F-scores for 8 out of 16 clonal TtgR variants after induction with naringenin. qRT-PCR fold enrichment data is presented as the mean ± SD of three biological replicates. The RNA-Seq fold enrichment value was calculated by summing the counts of all barcodes associated with a particular variant (see “Methods”). The R² for this dataset is 0.83. g Bootstrap correlation of qRT-PCR fold enrichment to RNA-Seq data for 8 of the 16 variants. Groups of 10, 25, 50, 100, or 500 barcodes were sampled for each variant across 500 cycles. Box plot elements same as (e). Source data are provided as a Source Data file.