Extended Data Fig. 10: High throughput assay to probe gene expression as a function of genomic position.

a. Graphical representation of the BacTRIP method. (I) A library of reporter plasmids generates a library of barcoded Caulobacter loci (purple, green, and red). Light gray: Tn5 mediated transposon. This includes a 5’ mosaic end (ME) followed by a SalI site, kanamycin resistance cassette (KanR), the origin of replication (ori), a promoter (gray), a gene of interest (yellow), a transcribed barcode (colors), a stop codon, the metK gene terminator from Caulobacter, and the 3’ ME. Transformation of the reporter plasmid library into Caulobacter introduces at most one barcode into each cell at a random locus (light brown box). Each barcoded strain reports local transcriptional activity at its locus. (II) DNA. Genomic DNA is used both to determine the integration locus (left) and the number of times each barcode was integrated (right). To determine the locus of each integrated barcode we digested the extracted DNA with SalI and used reverse PCR with mapping primers (Supplementary Table 8) to amplify fragments with an ori element for sequencing. To determine the number of times each barcode was integrated we amplified and sequenced the extracted DNA with counting primers (Supplementary Table 8). (III) RNA. We produced cDNA for sequencing from extracted, reverse-transcribed mRNA. The resulting quantity of inferred mRNA abundance was normalized by total copies of the barcode present in the library. b. BacTRIP results. We used three different CtrA binding sites to probe the wildtype CCNA_00350 promoter (PWT350), an engineered promoter in which we replaced the CtrA binding motif (red) of P350 with a consensus motif (PAT350), as well as a mutant site that is not recognized by CtrA (PMT350). We synchronized the three populations of cells and preformed BacTRIP on predivisional cells at the 90 minute time point. The bar graphs show abundance of 161, 156, and 148 barcoded eyfp as a function of locus distance from the new pole for pTripTn5- PWT350::eyfp, pTripTn5- PAT350::eyfp, and pTripTn5- PMT350::eyfp.