The genetic background strain for this work was MG1655 (CGSC #6300), obtained from the Coli Genetic Stock Center (CGSC). A commonly used alternate stock of MG1655 (seq) (CGSC #7740) harbors an IS1 element insertion in the regulatory region of FlhDC (53). CGSC #6300 is the original isolate of MG1655 submitted to the CGSC by the Blattner laboratory, which does not have any IS elements in the regulatory region of FlhDC. See the “Impact of insertion element mutation on flagellar transcription” section in the Supplementary Materials.

A cassette constitutively expressing mCherry was inserted into the IntS locus to improve identification of cells in flow cytometry experiments and facilitate segmentation of cells in time-lapse experiments. An expression cassette containing (i) a Zeocin resistance marker, (ii) an RNA-I promoter from ColE1 fused to a coding sequence for mCherry, and (iii) two 100–base pair (bp)–long flanking sequences homologous to the IntS gene was ordered from IDT as a gBlock. This linear fragment was chromosomally inserted into the IntS locus via chromosomal engineering techniques described above.

For all strains used in time-lapse experiments, the scarless technique described above was used to introduce an E98K point mutation in MotA (54), which disables flagellar rotation and prevents cells from swimming out of the channels in the “mother machine.” Flow cytometry was used to confirm that this point mutation did not affect the pattern of flagellar gene expression. Lists of plasmids and strains are available in tables S1 and S2.

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