Fig. 1: Identifying optimal spacing for repressors at lacUV5 promoter.
a We designed a library of lacUV5 variants modeled after the WT lacZYA promoter. In this library, we evaluate repressor effects when the distal site is moved 32 nucleotides upstream at 1 bp increments. b If repressors bind along the same face of the DNA helix, repression loop formation may occur, thereby preventing RNAP association with the promoter. c In this MPRA format, pooled promoter variants are engineered to express uniquely barcoded sfGFP transcripts, singly integrated into the essQ-cspB locus of the E. coli genome. After integration, individual promoter expression was determined en masse using the ratio of the barcode reads from RNA-seq to that of DNA-seq. d Comparison of MPRA expression measurements between biological replicates grown in MOPS rich-defined medium supplemented with 0.2% glucose (r = 0.987, P < 2.2 × 10−16, two-sided Student’s t test). e MPRA expression when a proximal site is added relative to expression of lacUV5 without repressor sites. Top shows the distribution of expression for all barcodes associated with each variant, whereas the bottom shows the averaged variant expression relative to lacUV5 without repressor site (null). Significance levels determined by Welch’s two-sided t test, ***P ≤ 0.001. AraC: n = 35, P = 0.07; GalR: n = 82, P = 6.68 × 10−15; LacI: n = 35, P = 2.22 × 10−7; LldR: n = 68, P = 0.47; PurR: P = 8.973 × 10−7. In each boxplot, the lower, middle, and upper hinges correspond to the first quartile, median, and third quartile, respectively. Whiskers represent 1.5× IQR from the lower and upper hinges. f Relative MPRA expression as each distal site is moved upstream in the absence of a proximal site relative to lacUV5 without repressors. Thick lines denote the fit using locally weighted polynomial regression. Thin lines connect data points at sequential intervals. Gray bars indicate 3 bp windows where the distal site is positioned in-phase with the +11 proximal site17. g MPRA expression as the distal site is moved upstream when the proximal site is present relative to expression of the proximal-only variant. Source data are available in the Source Data file.
