Labelling of chromosomal DNA in E. coli

The E. coli strain MG1655 was inoculated from glycerol stocks and grown overnight in lysogeny broth (LB) medium (Sigma) at 32 °C, while shaking at 200 r.p.m. Working cultures were inoculated 1:200 in lysogeny broth medium from overnight. 5-Ethynyl-2′-deoxyuridine (EdU) (10 μM; Baseclick) was added for 40 min at an OD₆₀₀ of ∼0.25, to cover the time needed for one complete replication round. As up to three or four replication rounds occur concomitantly at fast growth rates (here: 27 min doubling time)³² and chromosome replication requires ∼40 min at doubling times shorter than 60 min (ref. ³³), 40 min of exposure to EdU are sufficient to label the entire chromosome, even if the primary replication round has already replicated most of the chromosome. After fixation with 1% formaldehyde in phosphate buffer, cells were centrifuged at 5,000 g for 5 min and washed twice in 100 mM phosphate buffer (pH 7.4). The click reaction was performed as published previously¹⁹. Cells were washed thrice with PBS (Sigma), centrifuged and kept as stock solution. A volume of 20 μl of 20% (v/v) of the E. coli stock solution in PBS was mixed with 55 μl of imaging buffer and 60 μl of this solution was used in a sealed chamber for the optical trapping and dSTORM experiments. For 3D-SIM imaging, the E. coli stock solution was additionally labelled with 3.3 μM Sytox Green (ThermoFisher) for 5 min. This cell solution was immediately mixed with an equal volume of mounting media consisting of Vectashield H-1000 (Vector Laboratories) with 8.6 mM each of methyl viologen (Aldrich) and ascorbic acid (Fluka), before being mounted onto a slide and sealed from the external environment.